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Inorganic carbon utilization and the role of phosphoenolpyruvate carboxykinase in the photosynthesis of the marine macroalga Udotea

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Title:
Inorganic carbon utilization and the role of phosphoenolpyruvate carboxykinase in the photosynthesis of the marine macroalga Udotea
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Burch, Beth Ann, 1963-
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English
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xiii, 135 leaves : ill. ; 29 cm.

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Subjects / Keywords:
Algae ( jstor )
Carbon ( jstor )
Carbon dioxide ( jstor )
Carboxylation ( jstor )
Decarboxylation ( jstor )
Enzymes ( jstor )
pH ( jstor )
Photosynthesis ( jstor )
Species ( jstor )
Thallus ( jstor )
Botany thesis Ph. D
Dissertations, Academic -- Botany -- UF
Photosynthesis ( lcsh )
Udotea ( lcsh )
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bibliography ( marcgt )
theses ( marcgt )
non-fiction ( marcgt )

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Thesis:
Thesis (Ph. D.)--University of Florida, 1993.
Bibliography:
Includes bibliographical references (leaves 122-132).
Additional Physical Form:
Also available online.
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Beth Ann Burch.

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University of Florida
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University of Florida
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Copyright Beth Ann Burch. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
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31280131 ( OCLC )
0030441832 ( ALEPH )

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INORGANIC CARBON UTILIZATION AND
THE ROLE OF PHOSPHOENOLPYRUVATE CARBOXYKINASE
IN THE PHOTOSYNTHESIS OF THE MARINE MACROALGA UDOTEA

















By

BETH ANN BURCH





























A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 1993




































To My Dear Husband, Greg

and

In Loving Memory of My Parents,

John and Doris Lilly

















ACKNOWLEDGMENTS

I wish to thank Dr. George Bowes, my major professor, for his support and encouragement throughout my graduate career. The helpful suggestions provided by the supervisory committee, Dr. Steve Davis, Dr. Richard Smith, Dr. Charlie Guy, and Dr. Charles Allen are gratefully acknowledged. A special note of appreciation to Dr. Steve Davis, who so graciously volunteered to take his personal boat to Crystal Bay at all times of year in order to collect some of the plant samples. I appreciate the suggestions and help with research methodologies given by Dr. Julia Reiskind during the course of my tenure in Dr. Bowes' laboratory. A thank you goes to Dr. Charles Guy for allowing me to use some of his laboratory equipment in experiments. I am grateful to the research staff of the laboratory of the Florida Keys Land and Sea Trust in Marathon, Florida for their help in collecting some of the plant samples and for use of the laboratory facilities. This research was funded by the United States Department of Agriculture, Science and Education Administration, Competitive Research Grants Office, Photosynthesis Program Grants 88-37130-3703 and 90-37130-5576.

I am indebted to my husband, Greg, for his enduring love and support through my completion of this research and dissertation.

















iii



















TABLE OF CONTENTS

Page

ACKNOWLEDGMENTS ................. ........................ iii

LIST OF TABLES........................................... vi

LIST OF FIGURES.......................................... viii

KEY TO ABBREVIATIONS..................................... x

ABSTRACT................. ................................ xii

CHAPTERS

1 GENERAL INTRODUCTION AND LITERATURE REVIEW........ 1

The Marine Macroalga Udotea....................... 1
Habitat and Morphology ....................... 1
Calcification................................ 2
Photosynthesis................................... 3
C3 Photosynthetic Category.................... 3
C4 Photosynthetic Category.................... 5
CAM Photosynthetic Category .................. 9
Marine Algal Photosynthesis .................. 10
Chlorophyta ............................ 12
Phaeophyta ............................. 16
Rhodophyta ............................. 20
Bacillariophyta........................ 22
Summary ................................ 22
Phosphoenolpyruvate Carboxykinase ................ 23
Algae................................................ 24
Higher Plants ................................. 25
Fungi........................................ 28
Yeast................................... 28
Anaerobe of rumens...................... 28
Bacteria............................................ 29
Protozoa..................................... 29
Platyhelminthes.................................... 30
Nematodes.................................... 30
Vertebrates........................................ 31
Chickens ............................... 31
Rats ................................... 33
Other mammals........................... 33

2 INORGANIC CARBON UTILIZATION IN THE MARINE
MACROALGA UDOTEA ................................. 36

Introduction .......................................... 36
Materials and Methods............................ 37
Materials.................................... 37
Gas Exchange Experiments ..................... 37
pH Drift Experiments ...... ................... 40
Carbonate Salts in the Algal Skeleton......... 41


iv










Page
Carbonic Anhydrase Assay ..................... 41
Thallus Surface pH............................ 42
Chlorophyll Determination .................... 42
Results................................................. 42
Discussion............................................... 57

3 ROLE OF PHOSPHOENOLPYRUVATE CARBOXYKINASE IN THE
PHOTOSYNTHESIS OF THE MARINE MACROALGA UDOTEA.... 71

Introduction .......................................... 71
Materials and Methods............................. 71
Materials.................................... 71
Enzyme Extraction............................. 72
Enzyme Purification .......................... 72
Enzyme Assays ................................ 73
Protein Assay................................ 74
Enzyme Storage Conditions .................... 74
Enzyme Kinetic Analyses ...................... 75
Results................................................. 76
Discussion............................................... 109


LIST OF REFERENCES....................................... 122

BIOGRAPHICAL SKETCH...................................... 135











































v


















LIST OF TABLES
Table Page
2-1 Proportion of Udotea dry weight composed of 43 carbonates. DWA refers to the dry weight remaining
after acid treatment.

2-2 Effect of 10 mM CaCl on Udotea photosynthesis at 45
pH 8.2. Time elapse during photosynthetic
measurement was 15 min. Values reported are the
mean of 2 measurements SD.
2-3 Final pH of three replicates of Udotea following a 47
pH drift experiment in 200 pmol quanta m-2 s-1 for 6
hours at 320C. Values reported are the mean of 3
replicates SD.

2-4 Comparison of photosynthetic rates for Udotea 50
thallus sections measured at pH 8.2 and 300 pmol
quanta m2 s-1 and the predicted rates dependent
upon the spontaneous rate of CO2 production. The
predicted rates were calculated according to
Johnson (76). Measured photosynthetic rates are
the mean of 4 replicates SD.
2-5 Measurement of hydration time for CO2 at 10-120C, 51
recorded as a pH change of 2 units. Values
reported were the mean of four or five replicates
SD. The net photosynthetic rate at 2 mM DIC for
the thalli examined for the presence of external CA
was 26.47 5.77 pmol 02 g-1 FW h-1.
2-6 Surface pH measurements of Udotea thalli under 300 56
pmol -2 s-1 irradiation (400-700 nm), except as
noted, at 250C. The incubation medium for each was
artificial seawater with 2 mM DIC added. The two
pH values reported for the water were at the
beginning and end of the experiment. Each set of values reported was for eight to ten measurements
on one piece of thallus. The tip values were
recorded at the natural apical end of the thalli.

2-7 Effect of oxygen concentration on photosynthesis in 58
Udotea at pH 8.2 and 300 pmol quanta m-2 s-1.
Values reported are the mean of 4 replicates SD.
3-1 K values for PEPCK activity in crude extracts of 77
Uotea thalli. The values were derived from EadieHofstee plots.
3-2 Effect of various nucleotides on crude PEPCK 79
activity. The carboxylation rate was 0.642 0.018
pmol NADH mg-1 protein min -1 with ADP, and the
decarboxylation rate was 0.299 0.021 pmol NADH
mg- protein min -1 with ATP.

vi










3-3 Isolation of PEPCK from Udotea. 84

3-4 Effect of storage conditions on purified PEPCK 85
carboxylation activity. The initial activity was
15.17 pmol NADH mg-1 pro min-.
3-5 Form of dissolved inorganic carbon utilized by 90
Udotea PEPCK for carboxylation. The reaction was
performed at 120C to slow the equilibration between
CO2 and HC03- in solution.
3-6 Km values for substrates of isolated PEPCK at 92
optimal pH for the reactions determined from EadieHofstee plots. The K values for CO2 and HC03 at
each pH were calculated based on the HendersonHasselbach equation using the pK of 6.36 for
carbonic acid in freshwater at 25C (146).
3-7 Effect of oxygen in the gas phase on the 93
carboxylation and decarboxylation activities of
isolated PEPCK at various DIC and OAA
concentrations.
3-8 Influence of metabolites on PEPCK reactions. All 97
reaction components were present at saturating
concentrations for each reaction, except the effect
of the metabolite on the reaction for a given
substrate was measured at subsaturating
concentrations of that particular substrate.
Inhibition is reported vs. 1 mM substrate
concentration for carboxylation and vs. 0.02 mM
substrate for decarboxylation (unless noted
differently).
3-9 Influence of various metabolites and MDH/NADH on 108
the rate of PEPCK carboxylation measured as 14CO2
uptake.



























vii

















LIST OF FIGURES
Figures Page
2-1 Examination of inorganic carbon substrate used by 46 Udotea for photosynthesis. DIC was
added as mostly HCO (I) with and without CA by
adding NaHCO3 solution to the reaction mixture.
Of the 5 mM DIC present, 4.49 mM was HCO and 0.03
mM was CO DIC was added as CO (0) with and
without CA at a concentration of 5 mM by acidifying
NaHCO3 solution with HC1 in a syringe prior to
adding it to the reaction mixture.
2-2 Measurement of photosynthesis in reaction mixtures 49
of pH 7.7 (0o), 8.2 (A), and 9.0 (D) with varying
concentrations of CO2.
2-3 Effect of 40 mM KI on photosynthetic rate of Udotea 53
thalli at pH 8.0. (0) control, (0) 40 mM KI added.
2-4 Effect of DIDS on photosynthetic rate of Udotea 54
thalli at pH 8.0. (0) control, (o) 0.24 mM DIDS
added.
2-5 Effect of vanadate on photosynthesis of Udotea 55
thalli. The vanadate solutions were
prepared according to three different methods, as
indicated in Materials and Methods. The pH of
each reaction mixture with the vanadate solutions
A, B, and C added, respectively, was different:
7.93 (U), 7.86 (ii), or 7.98 (0).
3-1 Effect of either Mn2+ (0) or Mg2+ ( ) or both ions 80
present together at equal concentrations (I) on
PEPCK carboxylation activity from crude extracts.
3-2 Elution profile of the proteins PEPCK (0), MDH (0), 81
and Rubisco (A) according to molecular weight from the Sephacryl S-300 column. Protein concentration
(0) was measured at wavelength 280 nm as the
fractions eluted from the column.
3-3 Elution profile of the proteins PEPCK (0), MDH (0), 83
and Rubisco (A) according to charges on the
molecules from the Sepharose CL-6B column.
Proteins were eluted with a 0 to 0.3 M KC1 gradient
(0). Protein concentration (0) was measured at
wavelength 280 nm as the fractions eluted from the
column.
3-4 Effect of pH on the carboxylation (0) and 86
decarboxylation (0) activities of purified PEPCK at
250C.




viii










3-5 Effect of temperature on purified PEPCK 88
carboxylation activity (0) at pH 6.8 and
decarboxylation activity (0) at pH 7.8.
3-6 Influence of DIC concentration on PEPCK 89
carboxylation activity at the carboxylation optimum pH of 6.8 (0) and the decarboxylation optimum pH of
7.8 (0o).
3-7 Competitive inhibition of PEPCK carboxylation 94
activity by 100 pM MPA with respect to PEP. (0) no
MPA present, (o) with MPA present.
3-8 Competitive inhibition of PEPCK decarboxylation 95
activity by 100 pM MPA with respect to OAA. (0) no
MPA present, (0) with MPA present.
3-9 Effect of of 4 mM malate on the Km(DIC) for PEPCK 98
carboxylation. (0) no malate present, (o) with
malate present.
3-10 The effect of 4 mM DHAP on Km(DIC) for PEPCK 99
carboxylation. (0) no DHAP present, (0) with DHAP
present.
3-11 Effect of 4 mM 3-PGA on PEPCK carboxylation with 100
respect to DIC concentration. (0) no 3-PGA
present, (0) with 3-PGA present.
3-12 Effect of 4 mM F6P on the K (OAA) for PEPCK 101
decarboxylation. (0) no F6% present, (o) with F6P
present.
3-13 Effect of 4 mM F-1,6-BP on the Km(PEP) for PEPCK 102
carboxylation. (0) no F-1,6-BP present, (o) with
F-1,6-BP present.
3-14 Effect of 4 mM F-1,6-BP on Km(DIC) for PEPCK 103
carboxylation. (0) no F-1,6-BP present, (0) with
F-1,6-BP present.
3-15 Effect of 4 mM PEP on the K (OAA) for PEPCK 104
decarboxylation. (0) no PEP present, (0) with PEP
present.
3-16 Effect of ADP on the K,(ATP) for PEPCK 105
decarboxylation. (0) no ADP present, (0) with ADP
present.
3-17 Effect of 4 mM ATP on the Km(ADP) for PEPCK 107
carboxylation. (0) no ATP present, (o) with ATP
present.
3-18 Proposed scheme for photosynthetic carbon 120
metabolism in Udotea. Carboxylation of PEP occurs
by PEPCK in the cytosol. The OAA produced is
rapidly reduced to malate by cytosolic MDH, thereby
pulling the PEPCK reaction in the carboxylation
direction. Malate moves to the chloroplast where
it is decarboxylated, and the released CO2 is
refixed by Rubisco. The acceptor molecule PEP is
regenerated in the chloroplast by pyruvate Pi dikinase. The PEP can then move back to the
cytosol to accept another CO2'


ix
















KEY TO ABBREVIATIONS

A absorbance ATPase adenosine triphosphate synthetase AZ acetazolamide Bicine-NaOH n,n-bis(2-hydroxyethyl)glycine-sodium hydroxide BSA bovine serum albumin CA carbonic anhydrase CAM Crassulacean acid metabolism cAMP cyclic adenosine monophosphate CHES-NaOH 2-(n-cyclohexylamino)ethanesulfonic acid-sodium hydroxide

Chl chlorophyll Ci inorganic carbon DBAZ dextran-bound acetazolamide DCMU 3-(3,4-dichlorophenyl)-1,l-dimethylurea DHAP dihydroxyacetone phosphate DIC dissolved inorganic carbon DIDS 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid

DTT dithiothreitol DWA dry weight after acid treatment EZ ethoxyzolamide F-1,6-BP fructose-1,6-bisphosphate F6P fructose-6-phosphate Hepes-NaOH n-(2-hydroxyethyl)piperazine-n'-(2ethanesulfonic acid-sodium hydroxide KO.5 apparent Michaelis-Menten constant LN2 liquid nitrogen MDH malate dehydrogenase


x









MES 2-(n-morpholino)ethanesulfonic acid MES-NaOH 2-(n-morpholino)ethanesulfonic acid-sodium hydroxide

MPA 3-mercaptopicolinic acid OAA oxaloacetate PCO photorespiratory carbon oxidation PCR photosynthetic carbon reduction PEP phosphoenolpyruvate PEPC phosphoenolpyruvate carboxylase PEPCK phosphoenolpyruvate carboxykinase 3-PGA 3-phosphoglycerate PIPES-NaOH piperazine-n,n'-bis(2-thanesulfonic acid)sodium hydroxide

PMSF phenylmethylsulfonyl fluoride pro protein PVP-40 polyvinylpyrrolidone with molecular weight of 40,000 kilodaltons

Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase RuBP riubulose-1,5-bisphosphate Tris-HC1 tris(hydroxymethyl)aminomethane-hydrochloric acid

Vmax maximum velocity pCi micro Curie























xi

















Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy INORGANIC CARBON UTILIZATION AND THE ROLE OF PHOSPHOENOLPYRUVATE CARBOXYKINASE
IN THE PHOTOSYNTHESIS OF THE MARINE MACROALGA UDOTEA By

Beth Ann Burch

December 1993

Chairman: George Bowes
Major Department: Botany

Udotea is a marine, green, siphonaceous macroalga with C4-like photosynthetic characteristics. At pH 8.2, 15 mM dissolved inorganic carbon (DIC) was required to saturate photosynthesis, which is 7-fold greater than the concentration in natural seawater. Carbon dioxide was the preferred DIC species for photosynthesis. Acidification of the thallus surface occurred in the light and the dark, which could produce CO2 for diffusion into the siphons. External carbonic anhydrase activity was lacking. In pH drift experiments, the highest final pH value attained was only 8.95, which is in the range typical of CO2-only users. However, some HC03- use was indicated by enhanced photosynthesis when the HC03- concentration was increased at a given CO2 concentration. Photosynthesis was inhibited 13 to 33% by 4,4'-diisothiocyanostilbene2,2'disulphonate and 34% by the plasma-membrane ATPase inhibitor vanadate, which also halted thallus acidification. Thus, ATP may have a role in the DIC uptake process. Calcium chloride was necessary for photosynthetic DIC uptake, but not 02 evolution. This is consistent with calcification by Udotea, as 50% of the dry weight of the alga was composed of carbonate salts.




xii

















The kinetics of isolated phosphoenolpyruvate carboxykinase (PEPCK) were analyzed to determine how this enzyme operates for carboxylation in Udotea photosynthesis. Unlike carboxylation by ribulose-1,5bisphosphate carboxylase/oxygenase, the PEPCK carboxylation reaction was 02 insensitive though CO2 was the substrate. The carboxylation pH optimum of 6.8 was lower than for decarboxylation, and at this pH, which is close to the probable pH of the cytosol, where PEPCK appears to be located, the carboxylation Vmax was nearly 2-fold greater than that of decarboxylation. For carboxylation, the Km values for CO2 phosphoenolpyruvate (PEP), and ADP were 2.1 mM, 1.7 mM, and 30 pM, respectively; for decarboxylation, the KM values for oxaloacetate (OAA) and ATP were 35 and 2 pM. Dihydroxyacetone phosphate, 3phosphoglycerate, and ATP inhibited carboxylation, while fructose-1,6bisphosphate stimulated it by lowering the Km(PEP) from 1.6 mM to 0.5 mM. Fructose-6-phosphate and CO2 inhibited decarboxylation. These observations raise the possibility of metabolite regulation of the carboxylation/decarboxylation ratio. Both reactions were competitively inhibited with respect to PEP and OAA by 3-mercaptopicolinic acid. The carboxylation rate was enhanced 6-fold in the presence of malate dehydrogenase isolated from Udotea.

















xiii

















CHAPTER 1
GENERAL INTRODUCTION AND LITERATURE REVIEW


The Marine Macroalga Udotea

Habitat and Morpholoqy

Udotea conglutingata (Ellis and Solander) Lamouroux and U.

flabellum (Ellis and Solander) Lamouroux used in this investigation are marine, green, calcified, macroalgae that belong to the Family Udoteaceae of the Order Caulerpales, Class Chlorophyceae and Division Chlorophyta (20, 153). These algae can be found in tropical to subtropical marine waters (21), growing in soft mud or calcareous sand, often among seagrasses (45). The thallus of Udotea is composed of intertwining siphons with cell walls of 0-1,3-xylan (21, 23), which exists in a hollow helix conformation (97). No cellulose was detected.

Thalli are heteroplastidic with photosynthetic chloroplasts and starch-storing amyloplasts (153). Chloroplasts of Udotea have lamellae with various numbers of thylakoids and a thylakoid-organizing body of concentric double membranes at one end (153). The accessory photosynthetic pigments siphonein and siphoxanthin are also found in members of this family (153). In adult thalli, amyloplasts are found further down in the thalli than the chloroplasts (24).

Sexual reproduction in members of the Udoteaceae seldom happens

(21), but when it does occur, diploid dioecious thalli produce haploid gametes in a holocarpic manner, where an entire siphon is converted from the vegetative state to one where the nuclear division products are haploid gametes (101, 153). Gametes are released into the water when the siphon bursts open at the tip, and thallus death then follows (21). Anisogamous germ cells of two opposite mating types fuse, producing a


1









2

diploid zygote, which then develops into a protosphere, which has starch-containing chloroplasts (21). Upon maturation, the new thallus that develops from the protosphere becomes heteroplastidic. Vegetative reproduction, which is the most frequent type of reproduction in Udotea, occurs when new thalli arise from a branched rhizoidal system (21).

These habit and reproductive features of Udotea indicate that it possesses a number of derived and highly derived characteristics. Although its evolutionary development was in a different direction from that which eventally led to the vascular land plants, Udotea is by no means a "primitive" alga; rather, it belongs to a group considered "most derived (153)."

Calcification

Udotea conglutinata and U. flabellum are calcified representatives of this genus. The calcification mechanism in algae is tied closely with photosynthesis, as no CaCO3 is deposited until the plant is photosynthetically active (22, 23, 24).

The mechanism of calcification in these two species differs

slightly in that U. conglutinata deposits aragonite crystals of CaCO3 intracellularly within organic layers of a sheath that begins at the cell wall and forms an outer wall of the siphons (19). Some deposits also occur in the intersiphonous areas of the thallus. In addition, crystals of calcium oxalate are deposited in the vacuole in a randomly oriented fashion (56).

In contrast, U. flabellum lacks calcium oxalate crystal deposits in its vacuoles (56). At the outer surface, the siphons are highly branched, forming a developed cortical surface (19). In this cortex region, CaCO3 is deposited between layers of the siphon wall, while away from the exterior surface, CaCO3 aragonite crystals are deposited in the inter-siphon spaces (19). Both primary and secondary crystal deposits occur in the cortex region, and this pattern is somewhat similar to what occurs in sheath calicification, exhibited in U. conglutinata (19).









3

Thus, U. flabellum exhibits both intra- and intercellular calcification

(19).

Regardless of where CaCO3 precipitation occurs, this mechanism requires an enclosed space where a high pH can be generated, in this case, by the removal of CO2 for use in photosynthetic carbon assimilation (23). It has been suggested that the Ca2+ for precipitation in calcareous macroalgae comes from an intracellular pool

(98). The trans-membrane Ca2+ transport may be achieved by an ATPase that exchanges 2 H+ per Ca2 thus helping to generate the external high pH needed for CaCO3 precipitation (98).

Photosynthesis

Terrestrial plants have been divided into four categories based on the biochemistry of their photosynthetic mechanism. These categories are C3, C4, C3-C4 intermediates, and Crassulacean acid metabolism (CAM). The categories do not follow taxonomic lines, as one genus contains representatives from three of the four photosynthetic categories. The C3-C4 intermediate category will not be discussed. 3 Photosynthetic Category

By far the vast majority of plants in the North American flora fits into the C3 category. The leaf mesophyll of plants in this group is differentiated into a palisade layer on the adaxial side and a spongy layer on the abaxial side. Chloroplasts are dispersed throughout the mesophyll layer and are usually lacking in the bundle sheath. The chloroplasts are all the same, i.e., the thylakoids are stacked into grana, have both photosystems I and II, and have the full complement of photosynthetic carbon reduction (PCR) cycle enzymes. Of the seed plants, both gymnosperms and angiosperms (including monocots and dicots) are represented, so vascular bundle arrangement varies somewhat. Stomates are found on both leaf surfaces, although usually more prevalent on the abaxial surface. Guard cells are functional and open









4

during the daytime to facilitate the exchange of C002, 02, and water vapor between the leaf and the surrounding atmosphere (51).

The C3 plants use the PCR cycle to fix atmospheric carbon into

carbohydrate. Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) is the predominant carboxylating enzyme, and it incorporates atmospheric CO2 directly into 3-phosphoglycerate (3-PGA). Because of the active site conformation of Rubisco, which allows either CO2 or 02 to react with ribulose 1,5-bisphosphate (RuBP), the enzyme also catalyzes the first step in photorespiration or the photorespiratory carbon oxidation (PCO) cycle. Rubisco must be activated by C002 and Mg2+ to be catalytically competent (32, 89, 92). Carbon dioxide, rather than HC03or CO32-, is the carbon species utilized for both activation and catalysis, and the activator and catalytic CO2 molecules are distinct according to kinetic and spectroscopic measurements (75, 89). A C02 molecule and Mg2+ cation must bind to each of the eight catalytic sites for Rubisco to be completely activated (81). Light affects Rubisco in two important ways: light induces changes in the chloroplast such as increasing the stromal pH from around 7 to approximately 8.2 (92) and increasing Mg2+ concentration in the stroma by about 3 mM (92). These changes increase enzyme activity (32) and activate Rubisco via Rubisco activase. The binding of C002 and Mg2+ to the catalytic sites of the enzyme is facilitated (129, 154).

In the PCO cycle, RuBP is oxidized to 3-PGA and 2-P glycolate, the latter of which is dephosphorylated in the chloroplast, and the resulting glycolate enters the peroxisome, where it is metabolized to glycine. Glycine then migrates to the mitochondrion where C002 is released in the conversion to serine (91). The PCO cycle decreases net carbon fixation by 20 to 40% (16) because 02 directly competes for C02 at the Rubisco active site. This photorespiration decreases the amount of RuBP available for carboxylation, and one third of the fixed CO2 is released. The C02 compensation point, the concentration of C002 at which









5

the net carbon exchange in the light is zero, is achieved when the CO2 released by the PCO cycle just balances that reduced by the PCR cycle. For terrestrial plants, the CO2 released in photorespiration is a large component of the CO2 compensation point, but the CO02 released by dark respiration is not. When the plants are placed into 1% 02, where photorespiration is all but eliminated, measurable respiratory C02 release is virtually zero (54). The operation of the PCO cycle causes the CO02 compensation points in C3 plants to be quite high, in the range of 30 to 70 L CO02 L-1 (16). Photorespiratory CO02 release is energetically expensive because CO02 must first be reduced to RuBP with NADPH and ATP produced in the light reactions before oxidation to glycolate can occur (90), and the process itself requires NADH and ATP to function. The release of NH3 and its subsequent refixation is also a costly process.

Evidence that both carboxylation and oxygenation occur at the same site includes 1) inhibition of the two processes by carboxyarabinitol 1,5-bisphosphate (similar in structure to the C6 intermediate formed immediately after carboxylation of RuBP), 2) activation of both functions to the same degree by CO02 and Mg2+(91), and 3) demonstration that Km(CO2) equals Ki(02) and Km(02) equals Ki(C02) (26). However, although the ratio of CO02 to oxygen is relatively constant at all temperatures, oxygenation is favored over carboxylation at the higher temperatures because CO02 is less soluble in water than 02 under these conditions (65, 86, 107), and Rubisco has a lower affinity for CO02 at higher temperatures (104, 132). Therefore, the optimum temperature for C3 photosynthesis ranges from 15 to 2500C (16). C Photosynthetic Category

In some terrestrial plants, an additional metabolic pathway has arisen which serves to concentrate CO02 in the vicinity of the Rubisco active site, thereby decreasing photorespiration (48). The origin of the C4 photosynthetic mechanism appears to be polyphyletic, and for









6

terrestrial plants it is found only in the more advanced angiosperms (115). It occurs most frequently in herbaceous plants including annual and perennial herbaceous monocots and dicots.

Plants in this photosynthetic category exhibit Kranz anatomy in which the bundle sheath surrounding the vascular tissue is distinctive and composed of large, closely packed cells that are larger than the mesophyll cells. Chloroplasts in the bundle sheath are arranged specifically in accord with the type of C4 metabolism the plant uses

(48). Plants of the NADP-malic enzyme type contain dimorphic chloroplasts (48). Those of the mesophyll cells have grana, pyruvate Pi dikinase, and relatively high levels of NADP-malic dehydrogenase, but lack Rubisco and have the enzymes of only the reductive phase of the PCR cycle. However, the Kranz cell chloroplasts lack grana but have Rubisco and all of the PCR cycle enzymes (48).

All C4 plants have two carboxylation reactions. In the first reaction, HC03- is the form of dissolved inorganic carbon (DIC) used. Carbonic anhydrase (CA) facilitates the equilibrium between this species and atmospheric C02. Phosphoenolpyruvate is carboxylated with HCO3- by phosphoenolpyruvate carboxylase (PEPC), resulting in the production of the C4 acid oxaloacetate (OAA). Oxaloacetate is either reduced to malate or transaminated to aspartate, which is then transported to the bundlesheath cells. Either malate is decarboxylated directly or aspartate is deaminated and reduced to malate, which is then decarboxylated by one of three enzymes (48).

In the NADP-malic enzyme subgroup of C04 plants, which includes Zea mays and Saccharum officinarum, OAA from carboxylation in the mesophyll cell cytosol is reduced to malate and then translocated to the bundle sheath cell chloroplasts, where the malate is oxidized and decarboxylated to pyruvate and C02. The pyruvate is transported back to the mesophyll cell chloroplasts, where it is phosphorylated to PEP by pyruvate Pi dikinase. The C02 is then refixed by Rubisco in the











chloroplasts of the bundle sheath cells. Chloroplasts in the bundle sheath cells of these species are agranal and deficient in photosystem II activity (48). They are located near the centrifugal wall of the bundle sheath cells. The predominant transport metabolites in this subgroup are malate and pyruvate (48).

In the NAD-malic enzyme subgroup, exemplified by Panicum miliaceum and Amaranthus retroflexus, OAA from PEPC carboxylation is transaminated to aspartate in the cytoplasm of the mesophyll cells and then transported to the mitochondria of bundle sheath cells. Aspartate is deaminated to OAA in the mitochondria and then reduced to malate, which is oxidized and decarboxylated to pyruvate and CO2. Pyruvate is transaminated to alanine in the bundle sheath cell cytosol. Alanine moves back into the mesophyll cell cytosol and is deaminated to pyruvate, which them moves into the chloroplasts, where phosphorylation occurs via pyruvate Pi dikinase to regenerate PEP. The liberated CO2 is refixed by Rubisco in the chloroplasts of bundle sheath cells. Chloroplasts of the bundle sheath cells in these species do have grana and are located in the centripetal position in the cells. These plants also have many more large mitochondria in the centripetal position of the bundle sheath cells compared to other groups. Here the major transport metabolites are aspartate and alanine (48).

Plants in the PEPCK subgroup, such as Panicum maximum and Chloris gayana, transaminate the OAA produced by PEPC to aspartate in the cytoplasm of mesophyll cells, and aspartate is then moved to the cytosol of bundle sheath cells. Here deamination produces OAA which is decarboxylated to pyruvate followed by ATP-dependent formation of PEP in the cytoplasm. The PEP is dephosphorylated to pyruvate, which is then transaminated to alanine. Alanine then moves into the cytosol of the mesophyll cells. Alanine is deaminated to pyruvate which enters the chloroplasts and is phosphorylated to PEP via pyruvate Pi dikinase. As above, the C02 is refixed by Rubisco in the bundle sheath cell









8

chloroplasts. These plants have granal chloroplasts toward the centrifugal position in the bundle sheath cells. The major transport metabolites in this subgroup are also aspartate and alanine (48).

In the C4 photosynthetic mechanism, less than 5% of the C02

assimilated from the atmosphere enters 3-PGA directly. Instead, the C02 that enters the PCR cycle comes almost exclusively from the decarboxylation of the organic acids; essentially Rubisco is isolated from the atmosphere (48). This arrangement is beneficial for the plant in that the C002 concentration is elevated or concentrated. Photosynthesis by C4 plants is independent of 02 concentrations ranging from 2 to 21% (115). Because the C002/02 ratio is increased in C4 plants, the limitations imposed by C002 and 02 solubility characteristics with increasing temperature are overcome (86), which has a role in elevating the temperature optimum in these species into the 30 to 470C range (16). The function of the elaborate C4 acid system is to concentrate CO02 and in doing so, essentially eliminates the photorespiratory release of C002. However, some 2-P glycolate does form, and there is carbon flux through the glycolate pathway (108). A measurable indicator of less C002 release from the leaf is the very low C002 compensation point measured for C4 plants, usually in the range from

0 to 10 pL CO02 L-1 (16). Dark respiration does not constitute any measurable component of the C002 compensation point, as evidenced by these low values. The C4 acid mechanism, then, is an effective means by which to reduce photorespiratory C002 release.

Although less C002 is photorespired in the C4 pathway, thereby

reducing energy loss in the form of NADPH and ATP used to produce RuBP and energize the PCO cycle, C4 plants do require an additional two or three ATP molecules to fix CO2 because of the last step of the sequence where pyruvate is converted to PEP by pyruvate Pi dikinase (48). Under conditions of higher temperature and irradiance, C4 plants have a major advantage over C03 plants in that C4 plants are not affected by the









9

decrease in CO02 solubility with increased temperature because of the C02 concentrating mechanism (48). However, under lower irradiance levels and temperature, C3 plants can match or even exceed the net carbon gain exhibited by C4 plants (48).

CAM Photosynthetic Category

Crassulacean acid metabolism can actually be considered a form of C4 photosynthesis. Atmospheric CO02 is converted to HC03- by CA and fixed by PEPC into OAA, which is then reduced to malate. Decarboxylation then occurs by either NAD-malic enzyme or PEPCK, and the liberated CO02 is fixed into carbohydrate by Rubisco. Obligate CAM species utilize the CAM pathway for carbon fixation all the time, while facultative CAM species can shift dramatically the physiology and biochemistry of carbon fixation from C3 to CAM mode. Water availability determines the photosynthetic metabolism; low availability induces CAM (144). These plants exhibit variable CO2 compensation points. During the early part of the day, during Phase III of the CAM cycle, in which the malate stored at night is decarboxylated, the CO02 compensation point is low because the decarboxylation concentrates CO02 in the vicinity of Rubisco. The CO02 level may rise to be greater than 1% or 10,000 ML L-1. Although the 02 concentration increases as photosynthesis proceeds, the favorable CO02/02 ratio limits photorespiration (144). In addition, the difference in Km(CO2) and Km(02) for Rubisco favors CO02 fixation. However, toward the end of the day, during Phase IV when all of the stored malate has been depleted, the stomates open, and the plant carries on C3 photosynthesis. The CO02 compensation point in this phase has been measured at 50 pL CO2 L-1, and a burst of CO02 results when plants in this phase are artificially placed in the dark; both characteristics point to an active photorespiration process. Although CAM species can photorespire, the CO2 released can serve as an internal carbon source for photosynthesis (111). But unlike C04 plants, CAM species lack Kranz anatomy (59). PEPC and Rubisco are located in the same cells, but their










10

activities are temporally separate; fixation by PEPC at night results in the storage of malate in the vacuole until daylight when malate transport and decarboxylation and subsequent fixation of the released CO2 by Rubisco begins (113). Plants with this photosynthetic pathway usually occur in arid habitats, where the mechanism aids in water conservation. However, Isoetes howellii, a submersed aquatic plant, also exhibits this type of carbon metabolism (83) as a means of overcoming the carbon limitations in its environment. Marine Algal Photosynthesis

In numerous marine macroalgae studied thus far, photosynthesis is characterized by 02-insensitive photosynthesis, low CO2 compensation points, and low photorespiration rates, but some species do not share these attributes (77). Such characteristics make the algae photosynthetically more similar to the terrestrial C4 plants than to the C3 species. This feature is intriguing in that the algae are considered to be "primitive" among plants. The C4 photosynthetic mechanism is considered "advanced," as it involves specialized anatomy and compartmentalized enzymes with ample shuffling of metabolites between cells. One wonders how these "primitive" plants can utilize such an "advanced" form of metabolism. In contrast, Raven and Osmund (119) have concluded that some subtidal macroalgae, including the green Codium fragile, and the browns Dictyota dichotoma, Padina vickersii, Sargassum filipendula, and Fucus vesiculosus, possess photosynthetic capacity that cannot be used during steady-state, light-saturated photosynthesis unless the inorganic carbon concentration is increased, a scenario reminiscent of the terrestrial C3 species. Thus, the inorganic carbon concentration in marine waters is not sufficient to saturate photosynthesis for some algae, just as the atmospheric concentration of C002 does not saturate photosynthesis in the terrestrial C3 species.

One of the challenges a plant faces in a marine environment is

obtaining inorganic carbon for photosynthesis. Because dissolved gases











diffuse about 10,000 times more slowly in water than they do in air

(25), a submersed plant must be able to overcome this physical limitation in its environment. A survey of marine macroalgae across the algal divisions indicates that the DIC concentration in seawater is too low to overcome the effects of 02 on photosynthesis unless the alga employs some sort of carbon concentrating mechanism (71). Such mechanisms may involve direct HC03- uptake or C4 acid metabolism.

With regard to the particular species of DIC utilized by an alga, one must consider the influence of the pH and salinity of seawater on the proportion of each of the inorganic carbon species. The pH of seawater is in the 8.0-8.2 range and the salinity around 35 parts per thousand. Under these conditions, with the water in equilibrium with the atmosphere, the predominant species is HC03-, present at a concentration of about 2 mM. Only 10 pM of the total carbon is CO2 (133). It has been determined that CO32- is not a photosynthetically active species, even when its relative proportion dramatically increases, as may occur in a rockpool containing photosynthesizing algae

(93), so further discussion will focus on only HC03- and CO2. The macroalgae examined show a range of affininities for HCO3- (94). A common method of assessing this ability is based on measuring the photosynthetic rate and comparing it to the rate expected based on the CO2 produced as a result of HC03- dehydration (77). If the observed photosynthetic rate exceeds the spontaneous CO02 supply rate, the species likely is using HCO3- in addition to CO2 (77). However, before a specific HC03- utilization mechanism can be hypothesized, a lack of external CA must be demonstrated. This enzyme located outside the plasma membrane facilitates the conversion of HCO3- to CO2 or vice versa, depending on the pH. The CO2 can diffuse across the membrane into the cell (77), while HC03- cannot because of the charge on the molecule. Based on this criterion, most marine macroalgae studied have been found to need HCO3- in addition to the CO2 spontaneously supplied









12

by dehydration to support the observed photosynthetic rate (6, 77), and the algae show a consistently high capacity for inorganic carbon transport (14).

The inorganic carbon disequilibrium technique can also be used to ascertain the species of inorganic carbon preferred by a plant. The plant is given either CO2 or HCO3- under conditions where the time for equilibrium to be established between the two carbon species is prolonged by low temperature. Based on whether photosynthesis occurs more rapidly in the presence of either CO2 or HC03-, this rate difference indicates which particular carbon species is preferred by that plant (77).

Of course, the enzymes involved in inorganic carbon utilization are a key component of the photosynthetic mechanism. In all macroalgae examined, Rubisco has been determined to be the primary carboxylase (11, 71). In addition, the variability in photosynthetic rates observed for the marine macroalgae correlates well with the measured carboxylase activity (14), even to the extent that the photosynthesis to carboxylase activity ratio is close to one. Rubisco activity plays a role in regulating the photosynthetic capacity where internal CO2 concentration is high and photorespiration is suppressed, but other factors also have a role in limiting the photosynthetic rate under such conditions (14). Chlorophyta

Nearly all green macroalgae examined to determine their

photosynthetic mechanism exhibit a C3-type pattern, where the initial and primary carboxylating enzyme is Rubisco (11, 71, 77, 123). Studies with 14CO2 have shown that the radioactivity first appears in 3-PGA, the first product of Rubisco carboxylation. Only later, during the chase time, does the 14C appear in the C4 organic acids malate and aspartate, thereby indicating that these compounds are produced later in the metabolic sequence rather than being early products that are rapidly turned over, losing the 14C to other molecules (123).









13

The green macrophyte Prasiola stipitata, which grows in the very high splash zone, has a low CO02 compensation point and the photosynthetic rate is saturated at the seawater concentration of inorganic carbon (118). External HC03 is used for photosynthesis and no CAM-like features occur (118).

One green macrophyte studied extensively is Ulva. Some species of this ubiquitous alga move HCO3- across the plasma membrane into the cytoplasm, which saturates photosynthesis at the inorganic carbon (Ci) concentration found in natural seawater (10). Despite a low CO2 compensation point and 02-insensitive photosynthesis, Ulva is biochemically a C3 plant. Activity of PEPC is detectable, but the level is just one-tenth that of Rubisco. The data from radioactive labeling studies corroborate this finding; the first compound labeled with 14C is 3-PGA, and the radioactivity subsequently appears in sugars (11). Thus, it is suggested that a carbon concentrating mechanism based on HC03 uptake is responsible for these features (11).

During photosynthesis, the entire surface of U. fasciata reaches pH 10; no zones of acidification such as occur in the green alga Chara were detected (12). For U. lactuca, no carbonic anhydrase activity could be measured external to the cytoplasm (12, 47). When the CA is inhibited by acetazolamide (AZ), CO02 from the unstirred layer cannot support the observed photosynthetic rate; thus, it is concluded that HC03 uptake occurs, although CO2 use at times cannot be excluded (12).

Therefore, HC03 is the preferred species transported in U. lactuca (13). The mechanism for this movement could be HCO3-/H+ antiport or HC03/OH symport (12, 47). It is hypothesized that HCO3 is transported across the plasma membrane and then converted intracellularly to CO02 which moves to the chloroplast and can then be used by Rubisco for catalysis. Whether the HC03 translocation occurs via facilitated diffusion as occurs in red blood cell membranes or by active transport remains to be determined (47). In U. fasciata, the









14

internal carbon concentration can reach 2.3-6.0 mM with the bulk concentration of the surrounding medium at 0.02-1.5 mM, which indicates that active Ci accumulation occurs (13). With a cytoplasmic pH of 7.2 and high internal CA activity, the internal CO2 concentration could be high enough to suppress photorespiration and saturate carboxylation, even though this Rubiso has Km(CO2) of 70 pM (13).

A slightly different mechanism for Ci uptake may occur in U.

rigida. This alga has both an external and internal CA (15). HCO3- is converted to CO2 outside the cells, and CO2 passes through the plasma membrane. Support for this mechanism comes from studies with dextranbound AZ (DBAZ), which inhibits only external CA. In the presence of DBAZ, photosynthesis is inhibited at pH 8.6 when HCO3- is virtually the only inorganic carbon source, but no inhibition occurs at pH 6.5, where ample CO2 is present (15). However, active HCO5- transport cannot be excluded based on these data (15). In addition, the external alkalinization of the thallus is inhibited in the presence of DBAZ. Ethoxyzolamide (EZ) also inhibits photosynthesis, at both pH 6.5 and pH

8.6; however, adding CO2 to the medium at pH 8.6 overcomes the inhibition (15). The internal CA plays an important role in the Ci utilization mechanism in this alga.

Photosynthesis in Codium decorticatum appears C3-like with 02sensitive photosynthesis and minimal PEPC and PEPCK activities (123). The high CO2 compensation point is also sensitive to 02. The DIC in natural seawater does not saturate photosynthesis, but saturation occurs at 5 mM DIC (123, 124). Both the cytoplasm and isolated chloroplasts have CA activity, and some of the chloroplast activity is associated with the membranes (123). There is also a report of extra-cellular CA activity in Codium (40). In addition, higher photosynthetic rates can be obtained with the addition of HCO3- to the photosynthetic medium, which indicates that the alga is able to use HCO3- (124), as is also reported for C. fragile (119).









15

The green microalga Stichococcus bacillaris has high CA activity and a high affininty for CO2. In a medium of pH 5.0, the cells can concentrate internal CO02 to a level 20 times greater than that in the bathing medium, and this concentrating does not occur at pH 8.3 (102). Therefore, Stichococcus is a C02-user, and it is suggested that this concentration is accomplished via an active CO02 transport mechanism (102).

A green macroalga with photosynthetic metabolism that stands in stark contrast to other green algae investigated is Udotea flabellum. However, U. flabellum exhibits 02-insensitive photosynthesis and a low CO2 compensation point, both of which suggest a low level of photorespiration (123), similar to a number of the green algae. A comparison of photosynthetic rates measured at pH 5.0 and 8.0 indicates that the rate is higher at the higher pH, which suggests that the alga is able to use HC03- for photosynthesis (123). At pH 5.0, EZ has a limited effect on photosynthesis, but at pH 8.0, the rate decreases 39%, which shows that CA activity has a role in Udotea photosynthesis (123).

Examining photosynthesis in this alga with H14CO3- and with and

without 3-mercaptopicolinic acid (MPA), an inhibitor of PEPCK, indicates that decreased label is found in the photorespiratory products when PEPCK is operating and that the C4 organic acids malate and aspartate are early products that rapidly lose their radioactivity (123). Such a mechanism is reminiscent of the metabolism of the terrestrial C4 species. However, in those plants, the initial carboxylation reaction is catalyzed by PEPC, and the activity of this enzyme in U. flabellum was barely detectable (123). PEPCK has substantial activity, though, such that it rivals Rubisco activity (123). Inhibiting the PEPCK activity with MPA causes an increase in the photorespiratory intermediates along with a decrease in the radioactivity found in malate and aspartate (123).









16

Carbonic anhydrase activity in this alga may be related to HCO3use, but it is not a component of the mechanism to overcome the 02 inhibition of photosynthesis. Rather, it appears that PEPCK activity may be the key to reduce photorespiration. In contrast to other algae, it is doubtful that the 0-carboxylation catalyzed by PEPCK functions for anapleurosis. Instead, the C4-like metabolism depends on active PEPCK because inhibiting the activity with MPA decreases the photosynthetic rate by 70%, increases the Warburg effect, reduces the C4 acid label by 66%, and increases label in the PCO cycle intermediates by 3-fold (122, 123). These effects of MPA on photosynthesis cannot be explained by an HC03- utilization mechanism in U. flabellum, nor is it likely that the effects of MPA place PEPCK in the role of simply refixing photorespired C02; the effects are too large for this to be the case (122). Also, in the absence of DIC, exogenously-supplied malate is able to stimulate photosynthesis, but if MPA is present, no such stimulation occurs (122).

Because Kranz anatomy associated with terrestrial C4 species is not present in Udotea, a C4-type mechanism would have to involve an organellar separation of events (122). This type of separation has been reported for the freshwater angiosperm Hydrilla verticillata, which also lacks Kranz anatomy (25, 27). A parallel function between PEPCK of Udotea and PEPC of terrestrial C04 species is suggested; the activities of other enzymes typically associated with C4 metabolism are present in Udotea at high enough levels that it is reasonable to assume that C4 photosynthetic biochemistry could occur (122, 123). Thus, for Udotea, the evidence appears quite convincing that a type of C04 biochemistry occurs which is dependent on PEPCK activity. However, this mechanism has not been reported for any other marine macroalgae. Phaeophyta

The photosynthetic characteristics of the brown algae investigated display a mixture of features. For example, Turbinaria turbinata and Lobophora variegata exhibit a Warburg effect on photosynthesis at 0.5









17

and 2.5 mM DIC, respectively (71). Although Turbinaria has a detectable malate pool, it does not appear to function in a CAM-like mechanism

(71). Another brown, Dilophus guineensis, has high PEPC and PEPCK acitivities, but Rubisco is the major carboxylating enzyme (71). Of the brown algae surveyed by Kremer and Kippers (85), the PCR cycle is the only light-dependent carboxylation system, and greater than 90% of the early photosynthetic products are phosphorylated compounds, with 3-PGA being predominant, thereby lending support to the assertion that these plants operate C3 photosynthesis (85). They suggest that when PEPCK activity is high, the enzyme functions for light-independent carbon fixation. In addition, no PEPC activity is reported for the species they examined (85).

However, in contrast to Turbinaria and Lobophora, the eulittoral brown algae appear C4-like in their photosynthetic features; low CO2 compensation points, and 02-insensitive photosynthesis (79, 141), which suggests that an inorganic carbon concentrating mechanism is present in these algae. In addition, photosynthesis is saturated with inorganic carbon at the concentration of 2 mM found in natural seawater (141), so these algae are able to photosynthesize faster than would be expected based on the dehydration rate of HC03 alone (79).

Some Fucaceae, of which Ascophyllum nodosum is a member, for example, have C4-like physiology but C3-like biochemistry. They demonstrate an active membrane transport of CO2 and/or HCO3 although the location of the transporter, whether at the plasma membrane or chloroplast membrane, remains to be determined (78, 141). A. nodosum has a H+ buffering system that is independent of the ion exchange in the cell wall (5). This alga also has a photosynthetic buffering capacity connected to the H+ buffer, which is an adaptation to the alga's growing in the intertidal region. These buffering systems enable the plant to produce 02 in excess of the inorganic carbon taken up for photoreduction

(5). A. nodosum has high PEPCK activity, which may be involved in









18

producing the pool of C02-exchanging organic acids that function as the photoreductant buffer (5) as a complement to or an integrated part of a CAM-like system (6). The benefit of these two buffering systems to the alga is that they allow some photosynthetic electron transport to be maintained, even when exogenous carbon is in short supply. These systems could afford protection against photoinhibition (5).

When CA is inhibited in A. nodosum, the CO2 compensation point

increases 25-fold and the apparent K0.5(CO2) for photosynthesis increases 8-fold, resulting in a shift of photosynthetic gas exchange characteristics from C4-like to C3-like (77). The rate of photosynthesis equals the rate of CO2 production and diffusion from the bulk phase when external CA is inhibited. Therefore, it appears likely that CO2 is the species assimilated, and the C4 physiological characteristics depend in part on the operation of an external CA (77).

Two other species hypothesized to have an active HCO3- uptake

mechanism are Fucus distichus and Pelvetiopsis limitata, which have no external CA, but have photosynthetic rates that exceed the photosynthetic rate possible based on spontaneous HCO3- dehydration alone (42). Some algae of the Laminariales, including Laminaria digitata, L. hyperborea, L. saccharina and Alaria esculenta, and some others of the Fucales, such as Halidrys siliquosa, have some C4-like gas exchange characteristics, but the CO2 compensation points are higher than found in the Fucaceae, yet all have measurable external CA activity (141). These species appear to be less avid "HC03- users," but what features they do exhibit are probably due to active inorganic carbon influx as part of a concentrating mechanism (141).

Both Fucus and Laminaria exhibit light-dependent external

alkalinization of the surrounding medium, and this process is inhibited by adding DCMU, a light reaction inhibitor that blocks electron flow between quinone and the B protein, to the systems at pH 8.0 but not at pH 6.0 (64). Species of both genera have an external CA, as AZ inhibits









19

the alkalinization at pH 8.0 (64). The proposed mechanism for Ci uptake is that an external CA converts HCO3 from the surrounding medium to CO2 which then moves into the cells for photosynthesis. It is not known whether CO2 movement occurs by diffusion only or whether an active uptake process is involved (64). The site of CO2 transport has yet to be determined; it may be at the plasma membrane and/or the chloroplast envelope. Carbon dioxide is pumped from the external medium into the cytosol by using energy from photosynthetic electron flow rather than from mitochondrial respiration (140).

Another study of Laminaria digitata and several other species of brown algae that are Ci-limited in natural seawater showed that when photosynthesis is saturated by red light, a blue light pulse stimulates the photosynthetic rate (55). The effect is hypothesized to involve an increase in the CO2 supply to the plant; whether this is an effect on the external CA or H+ efflux pump in the plasma membrane or on the carbon-fixing enzymes in the chloroplast remains to be determined (55).

Photosynthesis in Dictyota dichotoma, Padina vickersiae, Sargassum filipendula, and Fucus vesicuolus can use HC03-, at least at pH 9.4 and

2 mM DIC (119). Of the four taxa, F. vesicuolus is able to take up HCO3 effectively and is carbon saturated at ambient concentration of DIC, similar to A. nodosum (77). Members of the Phaeophyceae are noted for exhibiting high PEPCK activity, but these organisms biochemically exhibit C3-like photosynthesis with 3-PGA appearing as the first product (85). Subsequent studies using 14C show a large amount of the radioactive carbon is located in malate, and then the malate is rapidly metabolized to mannitol (85). In F. vesicuolus, a substantial portion of 14C appears in malate and aspartate, thus suggesting active 0-carboxylation. However, rapid turnover of these organic acids does not occur, indicating that these compounds are really metabolic endproducts (85). In addition, Rubisco from A. nodosum has a carboxylase/oxygenase activity ratio of about six, which is similar to









20

that found in higher plants with C3 photosynthesis (77). For a long time, the PEPCK enzyme has been said to function in light-independent carbon fixation. However, in A. nodosum, dark fixation of carbon does not exceed 5% of the light fixation rate, and usually the dark fixation rate is much lower (77). Thus, the role of PEPCK in this group remains unclear, but it is suggested that DIC accumulation accounts for the observed gas exchange features that make some algae in this group appear C4-like in their metabolism (77). It is currently believed that a biochemically based C4-like photosynthetic metabolism is not widespread among the algae (77).

Rhodophyta

An investigation of photosynthesis in the red algae indicates that more than 90% of the early photosynthetic products are phosphorylated compounds, with 3-PGA constituting the largest proportion (85). These data support Rubisco as the major carboxylating enzyme, suggesting that the red alga incorporate inorganic carbon by the C3 mechanism (85).

In Laurencia papillosa, photosynthesis is inhibited by 02 at 0.5 mM DIC (71). However, the algal extract has high PEPC and PEPCK activities. Malate pools can be detected with levels greater at night than during the day, but the fluctuation is not large enough to imply the operation of a CAM mechanism (71). Thus, this alga probably operates a C3-type photosynthetic mechanism.

Photosynthesis in Chondrus crispus whole thalli, thalli pieces,

and protoplasts has been examined (136, 137, 138), and all the data are in agreement. The photosynthetic rate saturates at 3-4 mM DIC, and inorganic carbon does not accumulate internally, according to measurements using the silicone oil centrifugation technique (137, 138). This alga has an external CA that functions to dehydrate HC03 to C02, and the CO2 then passes across the plasma membrane (137, 138). Carbonic anhydrase activity is important for the function of this mechanism, as the initial carbon fixation rate decreases when CA is inhibited (137).









21

The CO2 translocation appears to be passive, since inhibitor studies do not suggest the presence of the band 3 anion transporter, a Na+/K+ transporter, or any involvement of Na+ in a transport mechanism (138). Therefore, internal accumulation of inorganic carbon cannot occur, as metabolic energy would have to be expended to do so. This alga can use HC03- in seawater via an indirect mechanism where a conversion to CO02 by CA occurs first, and then CO02 moves into the algal cells. Intracellular CA also plays an important role in this carbon utilization mechanism (138).

Another red macroagla, Gracilaria conferta, exhibits 02insensitive photosynthetic rates and a low CO02 compensation point under ambient DIC conditions (73). Rubisco is the major carboxylating enzyme, although PEPC and PEPCK activities are measurable. Inorganic carbon fixation occurs via the PCR cycle and supporting evidence for photosynthetic C4 acid metabolism is lacking (73). Photosynthesis saturates at close to ambient DIC conditions and HC03- is believed to be the main source of inorganic carbon. However, it is not known whether active uptake of HC03- occurs or if dehydration to CO02 occurs prior to uptake (73).

In Gracilaria tenuistipitata, an indirect HCO3- utilization

mechanism based on dehydration of HC03- to CO02 by an external CA has been proposed (63), similar to the mechanism hypothesized for Chondrus crispus. Carbonic anhydrase activity has been measured extracellularly and intracellularly, including an association with the chloroplast membranes (63). During photosynthesis, alkalinization of the surrounding medium depends on the Ci concentration and is inhibited by an inhibitor of external CA (63). However, the photosynthetic rate for thalli and protoplasts is higher at pH 6.5 as compared to pH 8.6, which indicates that CO02 is the preferred Ci species. At alkaline pH, the photosynthetic rate exceeds that which could be attained based on spontaneous HC03- dehydration alone, and when the external CA is









22

inhibited, the photosynthetic rate drops to that which would be expected based on the spontaneous dehydration of HC03 to CO2 (63).

In the presence of HC03-, Porphyridium purpureum has an

intracellular pH of 7.3, but when the cells are carbon starved, the pH falls to 6.0. Ethoxyzolamide blocks HC03--dependent alkalinization of the cytosol, so it is believed that CA and HC03 are important for cytosolic pH regulation (102). As is true for any enzyme, the activity of CA is pH-dependent, so a synergistic interaction may occur between CO2 uptake and HC03 transport (102). Li+ also inhibits HCO3--dependent photosynthesis in Porphyridium.

Bacillariophyta

An investigation of photosynthesis in the diatom Phaeodactylum tricornutum indicates that a HC03 transporter is present that depends on Na+ for its operation, as Li+ inhibits the HC03--dependent photosynthetic 02 evolution (46, 102). It is hypothesized that the Na+/HCO3 symport in air-grown and 5% C02-grown cells at the plasma membrane is driven by Na+/H+ antiport (46). Carbonic anhydrase plays an important role in converting the HC03 transported into the cells into CO2 for use by Rubisco (46, 102). Inhibition of CA by AZ at pH 8.0 increases the apparent K0.5(CO2) 2.5-fold, but inhibition by EZ increases

the K0.5(CO2) 10-fold (46).

Summary

The marine algae display a range of photosynthetic

characteristics. Some have very C3-like gas exchange features in having photosynthesis that is inhibited by 02, high CO2 compensation points, and photorespiration. Others display C4-like gas exchange characteristics, where the photosynthetic rate is not inhibited by 02, the CO2 compensation points are low, and photorespiration is low. Despite these features, in all algae examined thus far, the primary carboxylating enzyme is Rubisco, which points to the operation of a C3like photosynthetic mechanism. The C4-like gas exchange features are









23

accounted for by inorganic carbon concentrating mechanisms that involve either HC03- uptake or a CO2 pump. The net result is to diminish oxygenation by Rubisco, which results in the appearance of C4-like photosynthesis. However, Udotea flabellum is unique in that substantial evidence exists that supports the operation of C4-like biochemistry based on the high activity of PEPCK found in this alga, and this biochemistry may account for the C4-like gas exchange characteristics of this alga.

Phosphoenolpyruvate Carboxykinase

Phosphoenolpyruvate carboxykinase was discovered in chicken liver in 1953 by Utter and Kurakaski and later found to be present in all forms of liver and kidney tissue, as well as mammary, muscle, and adipose tissue (152). Utter also worked with pyruvate carboxylase, and between these two enzymes, the mechanistic basis for the entry of carbon into gluconeogenesis was established (69), as these enzymes function to bypass the step in glycolysis catalyzed by pyruvate kinase (69). In addition, this enzyme is present in a variety of other organisms: plants, algae, bacteria, fungi, yeasts, flatworms, roundworms, insects, and molluscs (152).

In most organisms, PEPCK catalyzes the synthesis of PEP from OAA as part of a pathway to synthesize carbohydrates according to the following reaction: OAA + ATP <===> PEP + C02 + ADP + Pi with Mn2+ as a cofactor (E.C. 4.1.1.49). Depending on the organism, other nucleotides that participate in the reaction include GTP (E.C. 4.1.1.32) or ITP. The reaction is readily reversible, but in general, the decarboxylation reaction is two to eight times faster than the carboxylation reaction (152). An exchange reaction between OAA and CO02 is also catalyzed by PEPCK and proceeds about 30 times more rapidly than the carboxylation reaction (152). The Mn2+ ions have two roles in catalysis: the ion binds directly to the enzyme in a one to one ratio and facilitates the









24

binding of PEP (152); it also forms a complex with the nucleotide species, and the complex is the catalytically active form (31, 33, 116). Algae

The green macroalga Udotea flabellum has PEPCK carboxylation

activity that equals the Rubisco activity (123). In this alga, PEPCK functions in the operation of a C4-like photosynthetic system that reduces photorespiration. Malate and aspartate appear as early photosynthetic products that turn over within one minute, and low amounts of radioactive label are present in photorespiratory compounds (123). This PEPCK enzyme is inhibited by incubation with MPA, and as a result, less malate and aspartate are produced as early photosynthetic products, and more photorespiratory intermediates are detected (122). Thus, the C4-like photosynthetic system and decreased photorespiration are linked to PEPCK activity hypothesized to be located in the cytosol (122).

The brown alga Laminaria hyperborea has PEPCK that uses ATP/ADP in catalysis. The enzyme functions for carboxylation in an anapleurotic role for replenishing the Krebs cycle to enhance growth in young fronds before light intensities are high enough for net photosynthesis to occur (159). The enzyme in Ascophyllum nodosum has a molecular weight of 60 kD and a pH optimum for carboxylation of 7.9 (84). In this species, PEPCK catalyzes light-independent carbon fixation with CO02 as the substrate (78).

The unicellular alga Euglena gracilis has high PEPCK activity when grown autotrophically with CO02 in the light. Higher activity is present when an additional carbon source, such as glucose, is supplied to the cells and no PEPCK activity is detectable in cells grown in CO2 in the dark (117). However, the highest activity occurs when the cells are grown in the dark with both CO2 and glucose. When Euglena is grown heterotrophically with lactate as the carbon source, PEPCK in the cytosol functions in gluconeogenesis to provide glucose for the cells









25

(117). PEPCK in this Euglena strain is suited to anaerobic CO02 fixation and is suggested to be the key enzyme for the methylmalonyl CoA pathway for odd-numbered fatty acids and alcohols, where propionyl CoA is incorporated into wax monoesters in fermenting Euglena (117). The enzyme is activated by a combination of Mn2+ and Mg2+, unlike other PEPCK enzymes. This PEPCK uses GTP/GDP for catalysis, and free sulfhydryl groups of the enzyme are essential for activity (117).

The marine diatom Skeletonema costatum has PEPCK that uses ADP that functions in O-carboxylation which is superimposed on the C3 photosynthetic pathway (104). Turnover of C4 acids does not occur and the Rubisco activity is always greater than that of PEPCK. Two pools of PEPCK may exist in this diatom: one pool is always activated, allowing for carboxylation in the light, while the second pool is inactivated in the light but activated in the dark. For this dark carboxylation, a substrate synthesized in the light, perhaps a polyol, is used as a source for PEP, thereby involving polyols in the primary metabolism of diatoms (104).

The diatom Phaeodactylum tricornutum has high PEPCK activity which has an absolute requirement for ADP and Mn2+ (72). The enzyme has a molecular weight of about 70 kD as determined by gel filtration and a pH optimum for carboxylation of 6.2 (72). This enzyme catalyzes carboxylation in vivo and uses HC03- as the inorganic carbon source. The carboxylation reaction may provide an additional source of ATP for the cells (72).

Higher Plants

In terrestrial plants, one subgroup of those with C4

photosynthesis has high PEPCK activity located in the bundle sheath cells, where the enzyme functions in the cytosol as a decarboxylase (156). In this subgroup, the PEPCK activity is 40 to 50 times greater than that in the other C4 subgroups or the C3 species (67).









26

Three well-studied species with this metabolism include Chloris guyana, Urochloa panicoides, and Panicum maximum (33). The PEPCK is a hexamer of 64 kD subunits with a total molecular weight of 380 kD and is active as a decarboxylase in the pH range from 7.4 to 8.2 (33). At pH 8.0, decarboxylation proceeds ten times faster than carboxylation in C. guyana (67). Although ATP/ADP is used in catalysis, the enzyme has a wide nucleotide specificity (33). The inorganic carbon species used as a substrate for the U. panicoides enzyme is CO2 (74). Decarboxylation activity of this PEPCK is inhibited by 3-PGA, fructose-6-P (F6P), fructose-1,6-bisphosphate (F-1,6-BP) and excess ATP but stimulated by Cl (33). Decarboxylation is also inhibited 40% by 0.5 mM CO2 (74). Actually, CO02 and HC03- inhibit PEPCK decarboxylation activity to nearly the same extent; this inhibition may be involved in dark regulation of the enzyme (74).

PEPCK activity in Chloris guyana has no pyruvate kinase activity associated with it (67). In contrast to PEPCK from chicken liver, this enzyme does not require the presence of a thiol-reducing compound for maximal activity. However, as for all other PEPCK enzymes, Mn2+ is essential for activity, and this ion cannot be replaced by any other divalent metal cation (33, 67, 151). The Mn2+ binds to the enzyme at its own site, and it also binds with the ATP as MnATP2-, as this species is the actual substrate for the enzyme (33). In the presence of Mn2+, Mg2+ inhibits the reaction (33).

In Spartina anglica, the levels of pyruvate, alanine, and 3-PGA cannot account for all the PEP produced by PEPCK in the C4 cycle (134). Either PEP moves directly from the bundle sheath cells to the mesophyll cells or more than one pathway of PEP metabolism is involved in this C4 cycle (134).

The reaction mechanism for PEPCK carboxylation in Panicum maximum is fully ordered with ADP binding to the enzyme first, followed by PEP and then C02. The OAA is released prior to ATP (2). However, the









27

enzyme from C. guyana has a fully random kinetic mechanism where C02, ADP, and PEP can bind in any order (2). Because CO2 can bind before the other substrates, the enzyme must have a CO2 binding site.

In the C4 species Urochloa panicoides, two decarboxylation systems are present in the bundle sheath cells. NAD malic enzyme decarboxylates malate in the mitochondria while PEPCK decarboxylates OAA in the cytosol (34, 66). Studies with radioisotopes indicate that PEP comes from aspartate via OAA while pyruvate comes from malate; 2-oxoglutarate is required in this system, presumably for OAA formation (34). The major source of ATP for PEPCK activity is phosphorylation in the mitochondria rather than photophosphorylation in the chloroplasts. For this system to work, malate has to be oxidized by NAD malic enzyme at one-third the rate that PEPCK operates to produce one NADH per three ATP utilized

(34). In the bundle sheath cells, mitochondrial electron transport contributes little to NADH oxidation; rather, NADH is oxidized primarily by reducing OAA to malate (66). In these mitochondria, Krebs cycle activity is not enhanced.

There is PEPCK in plants other than grasses. In apple, for

example, PEPCK activity peaks at the pre-climacteric stage and shortly after the fall of the petals; its role is to metabolize organic acids

(17). In other species with C3 photosynthesis, such as orange, lemon, avocado, and grape, PEPCK functions in the fruits to fix CO2 in the dark

(17).

The carboxylation and decarboxylation reactions of Panicum PEPCK are inhibited completely by 100 MM MPA while the exchange reaction is inhibited to a lesser extent (120). The inhibitor MPA at 100 pM concentration did not affect the activity of Rubisco or NAD-malate dehydrogenase (MDH), and NAD and NADP malic enzyme are only partially inhibited (120). The activity of PEPC is stimulated 70% by 1 mM MPA (120). Thus, in Panicum, MPA substantially inhibits only PEPCK. However, in Spartina, MPA is not a specific inhibitor of PEPCK because









28

HC03--dependent 02 evolution by bundle sheath strands was inhibited at pH 6.0, but not at pH 8.0 by MPA (134). FunQi

Yeast

In yeast, Saccharomyces cerevisiae, PEPCK is a tetramer (152) with one binding site per subunit (50) that utilizes the ATP/ADP nucleotides for reaction. The optimum pH for enzyme stability and catalysis is 6.5 (147). The molecular weight per subunit is about 64 kD with a holoenzyme molecular weight of approximately 261 kD (147). The enzyme functions in gluconeogenesis, but if a glucose source is available to the yeast, catabolite repression occurs, where PEPCK is degraded more rapidly in the presence of the glucose because the enzyme is not needed to make more glucose (106, 160). This catabolite repression is in contrast to Euglena, where PEPCK is synthesized in the presence of glucose (117).

Gluconeogenesis must occur in a respiring yeast cell, as the

cytochrome system and protein synthesis in mitochondria are necessary for the pathway to function (160). The catalytic sites on the enzyme contain active thiols, and each subunit has 10 cysteines that must be in the reduced form for the enzyme to be active (36). There is a Mn2+ binding site in addition to the site where ATP/ADP-Mn2+ binds. When ATP/ADP-Mn2+ is bound to the active site, it affords protection against inactivation of the essential arginine residues of the site (36, 95). Anaerobe of rumens

An anaerobic fungus of rumens, Neocallimastix frontalis, has a

PEPCK enzyme that is 608 amino acids long (125). The catalytic regions, with the sulfide binding site and and nucleotide binding domain, are highly conserved among fungal organisms and animals; however, the amino acid sequence of the yeast enzyme bears no similarity to the sequence of the rat, chicken, or fruit fly (125). Thus, although the enzymes may catalyze similar reactions, the proteins themselves have various










29

structures. It should be noted that the yeast enzyme utilizes ATP/ADP while the other enzymes mentioned above use GTP/GDP. Bacteria

In Veillonella parvula, an anaerobic bacterium of the human oral cavity, PEPCK functions in gluconeogenesis as a roundabout way of synthesizing PEP during lactate metabolism (38). The photosynthetic bacterium, Rhodospirillum rubrum, has PEPCK that operates as a carboxylase with CO2 as substrate (44). Bacteroides fragilis, a bacterium of the gut, has PEPCK that acts when low CO2 concentrations are present. It refixes the CO2 released from the decarboxylation of succinate to propionate to allow more ATP to be synthesized during glucose catabolism (37).

Protozoa

In Trypanosoma cruzi, the organism that causes sleeping sickness in humans, the PEPCK is located in membrane-bound glycosomes. The enzyme has a molecular weight of 84 kD and is composed of two subunits containing cysteine residues that are important for catalytic activity (150). The primary function of the enzyme is thought to facilitate the reoxidation of NADH from glycolysis by producing OAA that can be reduced to malate, and it also is involved in producing succinate, an end product of glucose metabolism (110). In addition, it functions to facilitate the complete oxidation of the amino acid skeletons that enter the Krebs cycle and operates in biosynthesis, functioning as a decarboxylase (150). Unlike PEPCK in yeast, this enzyme is not subject to repression by glucose (150).

In Leishmania mexicana, a blood fluke, PEPCK is also located in glycosomes, which are unique to the trypanosomatids (105). This enzyme catalyzes CO2 fixation and operates at a key branchpoint in the metabolism of Leishmania with PEP. High activity of MDH when PEPCK activity is also high, in the amastigote stage, may work to bring about succinate production in conjunction with fumarate reductase (105).









30

Platyhelminthes

The filiarial worm Setaria digitata, a facultative anaerobe, uses PEPCK to fix CO2 and ferment lactate (8). This enzyme utilizes ATP/ADP for catalysis. The PEPCK in the liver fluke Fasciola hepatica operates for reverse gluconeogenesis at the branchpoint of PEP to control the ratio of fermentation products by competing with pyruvate kinase for PEP

(88). Catalysis by PEPCK results in the production of acetate and propionate, while pyruvate kinase activity leads to lactate production

(88). Thus, the PEPCK functions as a carboxylase in an anaerobic environment using IDP (88). The enzyme is evenly distributed between the cytosol and the mitochondria, has an optimum pH range of 5.7 to 6.7, and has an absolute requirement for Mn2+ for catalysis. The activity of this PEPCK appears to be modulated only by the reaction substrate and product concentrations, and MPA is a potent inhibitor of catalysis (88). After OAA is produced by PEPCK, it is rapidly converted to malate by an active malate dehydrogenase, and the malate moves to the mitochondria

(88).

Nematodes

In the helminth Ascaris suum, PEPCK functions as a glycolytic C02fixing enzyme and is the point at which the metabolisms of this parasite and its host diverge (116). This enzyme is functional as a monomer with a molecular weight of about 80 kD and utilizes GTP/GDP for catalysis (116). As for other PEPCK proteins, Mn2+ binds directly to the enzyme to activate it. The active nucleotide complex can be formed with either Mn2+ or Mg2+ (116).

In the nematode Trichinella spiralis, PEPCK functions as a

carboxylase in the fumarate reductase pathway for the production of nvalerate, acetate, and propionate (18). This enzyme uses ITP/IDP with an optimum pH of 6.6, and 98% of the enzyme is located in the cytosol with the other 2% in the mitochondria (18).









31

Vertebrates

Many of the investigations on PEPCK have been performed with

chicken and rat tissue. In animals, PEPCK functions in liver and kidney tissue for gluconeogenesis, and since it catalyzes the first committed step of the pathway, it is also the rate-determining enzyme for the metabolic sequence that produces glucose from OAA (69). In gluconeogenesis, PEPCK and pyruvate carboxylase by-pass the glycolytic step catalyzed by pyruvate kinase. Within the livers and kidneys of humans, cows, sheep, cats, dogs, and guinea pigs, PEPCK is evenly distributed between the cytosol and mitochondria. In rats, mice, and hamsters, a larger proportion of PEPCK activity is found in the cytosol. In the livers of chickens, pigeons, and rabbits, more PEPCK activity is found in the mitochondria, but the distribution in the kidney is fairly even between the mitochondria and the cytosol (69). Chickens

This PEPCK is a monomer with a molecular weight of 70 kD, composed of 622 amino acids (43). The nucleotide used in the reaction is GTP/GDP (130). In the mitochondrial enzyme from embryo liver, the enzyme is activated by micromolar concentrations of Mn2+ in the presence of millimolar concentrations of Mg2+ (131). The OAA-CO2 exchange reaction is activated synergistically by Mn2+ and Mg2+ (131).

The regulation of PEPCK is different in birds and mammals in that avian livers are unable to synthesize glucose from pyruvate or amino acids (69). However, chickens have a high glucose concentration in the blood, and avian kidney tubules are able to use both pyruvate and lactate. In liver, lactate is the major precursor for glucose synthesis

(69). Gluconeogenesis from pyruvate and amino acids requires transfer of reducing equivalents and carbon from the mitochondria to the cytosol. In tissue with a cytoplasmic PEPCK, malate from the mitochondria moves to the cytosol, where it is oxidized by MDH to OAA, thereby producing NADH (69).









32

In contrast, in tissues that have very low cytosolic PEPCK

activity, such as chicken liver, the cytosolic NADH arises from the transformation of lactate to pyruvate in the cytosol. The low level of cytosolic PEPCK activity in chicken liver cells is attributed to posttranslational regulation, as considerable levels of PEPCK mRNA are present (157). Thus, the cytosolic enzyme in the kidney in a starvation state uses gluconeogenesis to convert the amino acids resulting from protein degradation into glucose (69). During starvation, the mRNA level for the cytosolic enzyme increases, after which PEPCK is synthesized (157).

The mitochondrial PEPCK functions as a part of the Cori cycle to oxidize lactate to pyruvate (69). The cytosolic and mitochondrial enzymes are synthesized from two different mRNA molecules (43), they exhibit subtle differences in ligand interactions at the active site, the proteins have different molecular weights, and they do not crossreact to antibodies produced from the other form of the enzyme (69). Another difference between the two enzymes is that the cytoplasmic form catalyzes the OAA-CO2 exchange reaction at a rate 30-100% greater than that catalyzed by the mitochondrial enzyme (130). The mitochondrial enzyme is synthesized in the cytoplasm as a precursor, having a signal peptide that is later excised after its arrival in the mitochondrion

(69). In fact, the cytosolic and mitochondrial forms from one species share only 56% homology in their amino acid sequences, while there are many similarities between the cytoplasmic enzyme from species from different classes, such as the chicken and rat (43, 69). Synthesis of the mitochondrial PEPCK is constitutive (43), while synthesis of the cytosolic enzyme increases in response to epinephrine, glucocorticoids, thyroid hormones, and glucagon which acts via cAMP (43, 69). Insulin inhibits cytosolic PEPCK synthesis.

In chicken liver cells, PEPCK is also found in the nucleus (145). The enzyme in this location functions for anapleurosis, in the nuclear









33

synthesis of N-acetyl-neuraminic acid, and the synthesis of glycosylated high mobility group proteins, which are non-histone proteins associated with the nuclear protein matrix (145). Rats

In rat liver cytosol, PEPCK functions in gluconeogenesis (149) and is a protein of 621 amino acids with a molecular weight of approximately 70 kD (9, 31). The enzyme utilizes GTP/GDP and is activated by micromolar concentrations of Mn2+ (31). The mRNA is about 2600 nucleotides long, and a portion of the gene favors Z-DNA formation (9). The hormone regulation of gluconeogenesis in rats and chickens is similar. Glucagon via cAMP and glucocorticoids stimulate PEPCK activity while insulin inhibits it (9). These hormones exert their effects by altering enzyme synthesis via mRNA amounts and translation (9). In rats that were starved and re-fed, serotonin increases the level of mRNA coding for the cytosolic form of PEPCK in the kidney, liver, and small intestine. The level is higher than found in the same tissues after 24 hours of starvation, and the serotonin stimulates cAMP levels within one minute of treatment (162), thus further affirming that production of PEPCK occurs in response to cAMP levels. Other Mammals

In camels, PEPCK functions in gluconeogenesis to maintain the high level of blood glucose found in these animals (1). More enzyme occurs in the kidney relative to the liver, but both isozymes have a molecular weight of 80 kD and pH optimum for activity of 7.2.

Rabbit PEPCK in the liver occurs in both the mitochondria and cytosol. The molecular weight of the cytosolic form is 68 kD, while that of the mitochondrial form is 56 kD as determined by gel filtration

(58). The cytoplasmic form functions in gluconeogenesis from amino acids, but the function of the mitochondrial enzyme is unclear. It may be involved in remobilizing lactate as a part of the Cori cycle (58).









34

Differences in molecular weight and function suggest differences in the amino acid sequence between the two forms (58).

In mouse pancreatic islets, the PEPCK uses ITP/IDP for catalysis and is activated by Mn2+ (68). Preincubation of the enzyme with ATPMginhibits the Mn2+-stimulated activity over time. Two fishes have been examined for their PEPCK activities. In the carp hepatopancreas, one form of PEPCK is present that has a molecular weight of 83 kD as determined by SDS electrophoresis and a pH optimum of 7.7 for decarboxylation and 7.2 for carboxylation (87). The liver of the rainbow trout has isozymes with molecular weights and pH optima similar to the carp enzyme (87). The enzymes function in gluconeogenesis. Of metal ions, Mn2 is the best activator (87). Summary

The PEPCK enzymes that utilize ADP/ATP for catalysis are generally either tetramers, as in yeast, or hexamers, as in the higher plants. The subunits typically have molecular weights in the 65 kD range. In contrast, the enzymes that use GDP/GTP for catalysis are monomers that have molecular weights in the 70 to 80 kD range. In most organisms where PEPCK is found, the most common location of the enzyme is in the cytosol, and the optimum pH for catalysis ranges from 6.2 to 7.9 among the organisms. For its reactions, PEPCK is Mn2+-specific. The ion binds to a site directly on the enzyme and may also form a nucleotide complex for the reactions. In organisms where PEPCK functions as a carboxylase, CO2 is the inorganic carbon substrate for the enzyme. In the algae, PEPCK functions as a carboxylase for anapleurosis and synthesis of fatty acids. In the higher plants, the enzyme operates as a decarboxylase in one of the C4 photosynthetic subgroups. In the vertebrates, PEPCK is a decarboxylase in the gluconeogenic pathway. Thus, this enzyme catalyzes a range of reactions for metabolism, and its activity in both directions is inhibited by MPA.









35

In Udotea, where PEPCK operates presumably in the cytosol, the

enzyme is hypothesized to function as an oligomer that uses ADP/ATP for its reactions. The Mn2+ ion also appears to be important for the reactions to proceed. This PEPCK activity appears to be crucial for the operation of the C4-like photosynthetic mechanism, as its inhibition with MPA shifts the photosynthetic metabolism to C3-like. Thus, Udotea PEPCK appears to share some features with PEPCK enzymes from some algae, yeast, and the higher plants. One of the purposes of this investigation was to learn more about the characteristics of PEPCK in Udotea to understand better how it plays a role in the C4-like photosynthetic mechanism of this alga.
















CHAPTER 2
INORGANIC CARBON UTILIZATION
IN THE MARINE MACROALGA UDOTEA

Introduction

Udotea, a marine siphonaceous macroalga of the Chlorophyta, has been found to exhibit C4-like photosynthetic characteristics, but the mechanism which produces these features requires more adequate investigation. C4-like gas exchange properties, such as 02-insensitive photosynthesis, low CO2 compensation point, and low photorespiration rate can be attributed to one of two general mechanisms: HC03utilization or C4 acid metabolism. It is evident that C4 acids are an important part of the photosynthetic metabolism of Udotea, as high activities of enzymes typically associated with this type of metabolism are found in this alga (123). In addition, during light fixation of 14CO2, there is an early appearance of C4 acids which turn over rapidly, suggesting that these acids are not just metabolic end-products (123). The purpose of this investigation was to determine if Udotea also uses HCO3 as a part of its photosynthetic carbon assimilation mechanism. Bicarbonate utilization could occur via an indirect mechanism, in which an external CA converts HC03 from the seawater into CO2 that either diffuses across the plasma membrane as proposed for the red alga Chondrus crispus (138) or be actively transported across, as reported for the green microalga Chlorella (126). Alternatively, could be used directly in a system where the ion is transported across the plasma membrane on a carrier which may or may not require energy to operate (135).

Since Udotea is a calcareous alga, the role of calcification during inorganic carbon asssimilation must also be considered, as



36









37

calcification and photosynthesis are closely linked in other calcifying organisms (24).

The results presented in this paper suggest that CO2 is the

primary species of inorganic carbon used in photosynthesis, but some energy-dependent HC03- use also occurs. A possible model for calcification is also considered.

Materials and Methods

Materials

Specimens of the marine macroalga Udotea conglutinata (Ellis and Solander) Lamouroux (division Chlorophyta) were collected with intact holdfasts in late fall, spring, and summer at the mouth of the Crystal River in the Gulf of Mexico, Florida, in water ranging from 1.0-1.5 m in depth. After collection, the plants were washed clean of epiphytes and transplanted into 20 L aquaria with mollusc shell fragments on the bottom, filled with filtered, aerated, natural seawater. The aquaria were placed into a growth chamber under a 250C/12-h photoperiod with a quantum irradiance of 5-10 pmol m-2 s-1 (400-700 nm) and a 230C scotoperiod. The seawater was changed every three to four weeks. Algae used for the investigations appeared healthy and had lush green color. Gas Exchange Experiments

Net photosynthetic rates were determined as 02 evolution with a Hansatech electrode system at 250C and 21% and 2% gas phase 02 (equivalent to 220 and 22 pM 02, respectively) in synthetic seawater buffered with 10 mM Tris-HCl, pH 7.7, 10 mM Tris-HC1, pH 8.2, or 10 mM CHES-NaOH, pH 9.0, containing various amounts of dissolved inorganic carbon, added as NaHCO3. The artificial seawater was prepared according to Drechsler and Beer (47) and contained 450 mM NaCl, 10 mM KC1, 10 mM CaC12, and 30 mM MgSO4. The salinity of this solution was determined by the HW Harvey method (96). A 10 mL sample of water to which four drops of 0.41 M K2CrO4 had been added was titrated with 0.16 M AgNO3 solution until a brick-red color persisted with stirring. The salinity, in parts









38

per thousand, was equivalent to the volume of AgNO3 solution needed to titrate the 10 mL sample. The quantum irradiance during the photosynthetic measurements was 300 pmol m-2 s-1 (400-700 nm). The rate of CO2 production in the photosynthetic medium arising from hydration and dehydration of the various inorganic carbon species present in seawater was calculated according to equation 6 from Johnson (76) using the constants determined for seawater with a salinity of 34 parts per thousand at a temperature of 250C.

The calculation for the hydration rate of CO2 in seawater is presented according to the method of Johnson (76).

6(CO2)/6t = -(KC02 + K0H KW/aH)(C02) = (kdaH + KHCO3_)(HCO3) M = mol L-1

For seawater at 250C and 34 parts per thousand salinity:

KcO2 = 0.0362 s-1

KOH KW = 13.4 x 10-11 mol L-1 s-1 Kd = 3.52 x 104 L mol-1 s-1 KHC03_ = 1.17 x 10-4 s-1 aH = [H+]

At pH 8.2 and 2 mM DIC:

[H+] = 6.31 x 10-9 M

[C02] = 0.00601(DIC) = 1.20 x 10-5 M [HC03-] = 0.898(DIC) = 0.001796 M Therefore, substituting into the equation:

6(C02)/5t = -[0.0362 + (13.4 x 10-11)/6.31 x 10-9](1.20 x 10-5)

+ [(3.52 x 104)(6.31 x 10-9) + 1.17 x 10-4 )](0.001796)

5(C02)/6t = -[0.0362 + 0.0212](1.20 x 10-5) = (3.39 x 10-4)(0.001796)

= -(0.0574)(1.20 x 10-5) + 6.09 x -7

= -(6.89 x 10-7) + (6.09 x 10-7)

= -8 x 10-8 s-1 x M = -0.08 x 10-6 s-I x M

=-4.8 x 10-6 min-1 x M









39

The effect of CaCl2 on photosynthesis was measured in artificial seawater prepared with and without CaCl2. Sections of thallus, about 15 mm by 15 mm, from the tip of the thalli, were incubated for 15 min in unbuffered, artificial seawater with or without 10 mM CaC12 at a quantum irradiance of 150 pmol m-2 s-1 (400-700 nm). The photosynthetic rate measurement in the presence of 2 or 5 mM DIC commenced with adding the alga to the oxygen electrode chamber, which already contained 2 mL artificial seawater buffered with 10 mM Tris-HC1, pH 8.2. Photosynthesis was measured for 15 min. An aliquot of the reaction mixture in the 02 electrode chamber was removed to measure the initial total inorganic carbon. A 100 ML sample was injected into 3 mL 0.1 N H3PO4 bubbled with N2 in a flask, and the CO2 released was measured with an infra-red gas analyzer and recorded on an integrator against a known concentration of a KHCO3 standard. Another aliquot of the reaction solution was removed after the photosynthesis measurement to record the total DIC remaining.

The influence of several potential effectors on photosynthesis was examined, including 40 mM KI, 0.24 mM DIDS, and 0.1 mM vanadate. The vanadate solutions were prepared according to three different methods. In the first method, solution A, 5 mM Na3VO4 was dissolved in 20 mM NaOH and allowed to stand overnight at room temperature (57). In the second method, solution B, Na3VO4 was dissolved in deionized water (20 mL) to produce a 5 mM solution and heated for 3 h at 400C (109). After cooling, the volume was readjusted to 20 mL with deionized water, and the pH was adjusted to 9.5 with MES powder. The absorbance of a 0.1 mM solution was recorded at 268 nm against a water blank in order to determine the true concentration of the solution. The molar extinction coefficient used was 1.44 x 10-4 as determined by Cantley et al. (35). The pH was then adjusted to 7.2 with MES powder and the solution was stored in a glass stoppered flask at room temperature until used. In the third method, solution C, the solution was prepared on the day of









40

the experiment as 100 mM solution in 20 mL deionized water (142). The pH was adjusted to 9.5 with MES powder, and the solution was boiled until colorless. After cooling, the pH was adjusted to 7.5 with MES powder, followed by a second boiling. After cooling, the volume was adjusted to 20 mL with deionized water and the pH adjusted to 7.5 with MES powder. Potential effectors were added into the standard reaction mixture, consisting of artificial seawater buffered with 10 mM Tris-HCl at pH 8.2, to which NaHCO3 was added to achieve various DIC concentrations.

The 02 electrode system was also used to determine which species of inorganic carbon was used for photosynthesis. These photosynthetic measurements were performed at 90C to slow the equilibration between CO2 and HC03-. The procedure that Cooper et al. (44) used to determine the DIC substrate of carboxylation enzymes was modified for use on thalli pieces. The Udotea thallus section was placed into a chamber containing

2 mL artificial seawater buffered with 10 mM Tris-HCl at pH 8.2, at a quantum irradiance of 300 pmol m-2 s-1 (400-700 nm). When a stable line was achieved on the chart recorder, either 5 mM NaHCO3 or 5 mM NaHCO3 acidified with 0.1 N HC1 was added to the chamber with a Hamilton syringe. If an acidified solution was used, the acidification was done in the syringe, and the solution was added to the reaction chamber only after bubbles of CO02 appeared in the syringe. In this manner, a significantly larger proportion of either CO02 or HC03- was added to the reaction chamber. The photosynthetic rate was followed for a maximum of one minute.

pH Drift Experiments

Udotea thalli without holdfasts were placed into 60 mL glassstoppered bottles containing unbuffered, artificial seawater to which 2 mM NaHCO3 was added (94). The pH of the starting solution was recorded and a sample was removed to measure the true concentration of total inorganic carbon present at the start of the experiment. The stoppered









41

bottles were placed into a shaking water bath maintained at 320C under a quantum irradiance of 200 pmol m-2 s-1 (400-700 nm) for 6 h. After removal from the water bath, the bottles were allowed to return to room temperature, at which time the final pH of the solution was measured. An aliquot of water was removed to measure the remaining total DIC, and the alkalinity of the water was measured by titrating a 10 mL sample of the water with 0.121 N HC1 to pH values of 4.2, 3.9, and 3.7 (94). The quantities of acid added and the exact pH values achieved were used to calculate the alkalinity of the samples. The Udotea thalli were removed and the fresh weights recorded. The thalli were then dried in a 7000 oven for at least 24 h and dry weights determined. The HCO3 dehydration rate was calculated using equation 6 and the constants from Johnson (76).

Carbonate Salts in the AlQal Skeleton

Thalli that had been oven-dried were placed into Petri dishes and treated with 6 N HCI to liberate the carbonates in the plant skeleton. After bubbling ceased, the acid was pipetted off and the remaining thalli allowed to dry. After drying, the weights were measured and compared to the fresh weights and the dry weights prior to acid treatment.

Carbonic Anhydrase Assay

Carbonic anhydrase was assayed according to the method of

Drechsler & Beer (47). At 10-120C, the time for a pH change of two units was recorded for a solution of 5.8 mL artificial seawater, 0.2 mL Tris-HC1, final concentration 10 mM, pH 8.6, to which 4 mL of ice-cold C02-saturated water was added. The time required to achieve the same pH change was also noted when 10 units of carbonic anhydrase or a piece of Udotea thallus were added. The thallus had been cut the previous day so that wound healing would occur (41) and the enzyme being measured would be located on the thallus, rather than having leaked from the cytosol of the alga.









42

Thallus Surface pH

Udotea thalli sections cut the night before experimentation were placed into Petri dishes containing 40 mL unbuffered artificial seawater with 2 mM NaHCO3 added. These sections were incubated in the growth chamber for 15 min, under a quantum irradiance of 5 pmol m-2 s-1, after which time the thalli were moved to a fresh solution of 2 mM NaHCO3 in artificial seawater. The thalli sections were then exposed to a quantum irradiance of 300 pmol m-2 s-1 (400-700 nm) while the pH of the thallus surface was recorded with a microelectrode (49). Measurements were made at various points on the thallus, and the change in the pH of the water from the beginning of the thallus measurements until the end was also recorded. Thallus measurements were also made in the presence of 40 mM KI, 0.24 mM DIDS, and 1 mM vanadate, as well as in the dark, and for the water in the absence of thalli.

Chlorophyll Determination

Chlorophyll was determined by the method of Arnon (3). The

thallus was ground at 40C in a mortar and pestle in 2 mL 10 mM Tris-HC1, pH 7.3. A 0.2 mL aliquot of the homogenate was pipetted into 0.8 mL 100% acetone. This solution was incubated on ice, in the dark. After 15 min, 2 mL 80% acetone was added to the solution, which was incubated for another 15 min period on ice in the dark. The solution was then centrifuged at 3600 g for 5 min at room temperature. The absorbances of the clear solution at 645 and 663 nm were determined against a blank of 80% acetone. The equation for the calculation is: [(A663 x 0.00802) + (A645 x 0.0202)] x 5 (dilution factor) x 2 (total mL in grind) = total mg Chl in thallus.

Results

An examination of the structural components of the Udotea thallus indicated that the fresh weight of the thallus was about double the dry weight, and nearly 50% of the dry weight was composed of carbonate salts that could be removed by treatment with HC1 (Table 2-1).










43









Table 2-1. Proportion of Udotea dry weight composed of carbonates. DWA refers to the dry weight remaining after acid treatment.

Carbonate
Component
Fresh Dry Dry Weight of
Thallus Weight Weight FW/DW After Acid FW/DWA Skeleton


g g %
1 0.97 0.44 2.20 0.26 3.73 41 2 1.59 0.75 2.12 0.36 4.42 52 3 1.72 0.73 2.36 0.40 4.30 45 4 1.07 0.49 2.18 0.25 4.28 49 5 1.03 0.50 2.06 0.25 4.12 50 Mean --- --- 2.18 --- 4.17 47
0.11 0.27 4









44

The total amount of 02 evolved during photosynthesis by Udotea at 2 mM DIC in the absence of CaCl2 was similar to that with CaC12 present, although with 5 mM DIC in the medium, 22% more 02 was evolved during the same time period when CaC12 was included in the photosynthetic reaction mixture (Table 2-2). However, a noticeable difference occurred with regard to the net change of the total inorganic carbon in the medium when CaC12 was omitted from the medium. The presence of the CaC12 seemed necessary for inorganic carbon to be removed from solution, as in its absence, more DIC was present at the end of the 15 min photosynthetic period than at its beginning. In the presence of CaCl2, the quantity of inorganic carbon removed by the thallus section in 5 mM DIC was 6-fold greater than the amount removed in 2 mM DIC. When CaC2 was absent, about twice the amount of inorganic carbon was present after photosynthesis in the chamber which started with 2 mM DIC as compared to the chamber with 5 mM DIC.

The species of inorganic carbon used for photosynthesis was

investigated (Fig. 2-1). By using a method modified from a study of Cooper et al. (44) on isolated enzymes, whole-thallus photosynthesis was carried out at 90C so as to slow the equilibration of the carbon species added to the reaction medium. The experiment was performed at pH 8.2. When DIC was added as virtually all C02, the photosynthetic rate was five-fold higher than when the DIC was added so that 90% of the carbon was present as HC03-. When CA was added along with HC03-, no stimulation of photosynthesis was observed. When CA was added with CO2, the photosynthetic rate dramatically decreased by five-fold, to the level attained in the presence of HCO3- alone.

In a pH drift experiment, where intact Udotea thalli were

incubated for six hours under a quantum irradiance of 200 pmol m-2 s(400-700 nm), the final pH attained by the plants was approximately 8.3 (Table 2-3). Of the total DIC present at the conclusion of the experiment, the CO2 component was 5 uM, while nearly 88% of the










45








Table 2-2. Effect of 10 mM CaC12 on Udotea photosynthesis at pH 8.2. Time elapsed during photosynthetic measurement was 15 min. Values reported are the mean of 2 measurements SD.

With Without With Without
CaCl2 CaC12 CaCl2 CaC12 DIC
Concentration Total 02 evolved Net Change in total DIC


mM mmol g-1 FW
2 0.11 0.03 0.10 0.06 -0.23 2.17 5 0.14 0.01 0.11 0.03 -1.30 1.10

Note: A positive value indicates that more inorganic carbon was present in the chamber after the photosynthesis measurement was made than at the start.



















25



20



LL 15



0 10
-5

5


0
+ CA + CA



Figure 2-1. Examination of inorganic carbon substrate used by Udotea for photosynthesis. DIC was added as mostly HC03 (U) with and without CA by adding NaHCO3 solution to the reaction mixture. Of the 5 mM DIC present, 4.49 mM was HC03 and 0.03 mM was COp. DIC was added as CO2 (0) with and without CA at a concentration of 5 mM by acidifying NaHCO3 solution with HC1 in a syringe prior to adding it to the reaction mixture.









47









Table 2-3. Final pH of three replicates of Udotea following a pH drift experiment in 200 pmol quanta m- s1 for 6 hours at 320C. Values reported are the mean of 3 replicates SD.

HCO3
Final Final Dehydration Mean Final pH Total DIC CO2 HC03- Rate


pM pmol min-1
8.26 0.40 1000 464 5 2 877 407 0.29









48

inorganic carbon was present as HC03-. At this pH, CO2 was produced from HC03- at the rate of 0.29 pmol min-1. In another pH drift experiment, where the amount of DIC present at the start of the incubation period was only that added as a result of bubbling the artificial seawater with air for 0.5 h, the final pH reached was 8.95

0.42 for 15 samples.

The manner in which pH affects photosynthesis was investigated (Fig. 2-2). For similar CO2 concentrations, the additional HC03available at higher pH values was able to increase photosynthesis. At pH 9.0, 0.06% of the total DIC is CO02 and 59.6% is HCO3-, at pH 8.2,

0.6% is CO2 and 89.8% HC03-, and at pH 7.7, 2.0% is CO2 and 94.8% HC03-. This higher photosynthetic rate suggested that some HCO3- was being employed for photosynthesis in addition to the CO2, although the preferred species was CO02 (Fig. 2-1). It can also be seen that even though 30 mM DIC was added at pH 8.2 and 50 mM DIC at pH 9.0, the photosynthetic rate still was not saturated due to the small proportion of inorganic carbon present as C02'

The rate of CO02 production from HC03- was calculated for this

system using the equation of Johnston (47) (Table 2-4). If the rate of photosynthesis was greater than the rate of spontaneous CO02 production from HC03-, then the evidence suggests that the additional photosynthetic rate was supported by HCO3- utilization (47). At both 1 and 2 mM DIC, the measured photosynthetic rate for Udotea was greater than the rate predicted from spontaneous HC03- dehydration (Table 2-4).

Udotea thalli were investigated to determine whether any external CA activity was present. The time for spontaneous HCO3- dehydration was 13 s, and adding 10 units of CA decreased the time by nearly 50% (Table 2-5). However, the presence of the Udotea thallus actually slowed the rate to 19 s, thus suggesting that no external CA was present on the thallus.



















30


25

-C

20




0 I I T






0 50 100 150 200 CO2 (.M)



Figure 2-2. Measurement of photosynthesis in reaction mixtures of pH 7.7 (o), 8.2 (A), and 9.0 (0) with varying concentrations of C02.









50








Table 2-4. Comparison of photosynthetic rates for Udotea thallus sections measured at pH 8.2 and 300 pmol quanta m-2 s-I and the predicted rates dependent upon the spontaneous rate of CO2 production. The predicted rates were calculated according to Johnson (76). Measured photosynthetic rates are the mean of 4 replicates SD.

Photosynthetic Rate

DIC O, Measured Predicted

mM % Pmol 02 h-I
1 21 0.37 0.08 0.29
2 0.57 0.24
2 21 0.44 0.10 0.58
2 0.78 0.07
5 21 0.80 0.25 1.46
2 1.18 0.17










51









Table 2-5. Measurement of hydration time for CO2 at 10-120C, recorded as a pH change of 2 units. Values reported were the mean of four or five replicates SD. The net photosynthetic rate at 2 mM DIC for the thalli examined for the presence of external CA was 26.47 5.77 mol 02 g-1 FW h-1.

Condition Time for Reaction sec
Spontaneous 13 3 With 10 units CA 8 1 With Udotea intact thallus 19 2









52

Udotea photosynthesis was examined in the presence of several potential effectors of HC03- use. In the presence of 40 mM KI, where the I- may interfere with HC03- uptake, photosynthesis was inhibited 26 to 35%, and the inhibition increased with increasing HCO3- concentration (Fig. 2-3). These measurements were made without any pre-incubation of the thallus in KI.

The photosynthetic rate was also inhibited in the presence of 0.24 mM DIDS, (Fig. 2-4), which inhibits transport by the band three anion transporter in red blood cells. The inhibition ranged from 13% at 3.7 mM HCO3 to 33% at 0.5 mM HC03-.

Vanadate halts ATP synthesis by interfering with phosphate binding at plasma membrane ATPases (35). Each bar in Fig. 2-5 represents four alga pieces incubated for a period of 2 h in 0.01 mM vanadate solutions prepared by different methods. Several solution preparations were used to determine whether the vanadate ion species present in a particular solution was more effective at inhibiting the ATPase, if it were present. Photosynthesis was inhibited only 3% after 5 min incubation in solution A (Fig. 2-5), but this inhibition increased to 34% inhibition after a 2 h incubation period. Incubation in solution B did not appear to inhibit photosynthesis of Udotea thalli. For thalli incubated in solution C, photosynthesis was inhibited 19% after just 5 min, but no further inhibition resulted after prolonged incubation.

Surface pH measurements of Udotea thalli exposed to a quatum

irradiance of 300 pmol m-2 s-1 were made on thallus pieces incubated in unbuffered, artificial seawater containing 2 mM DIC at 25 to 300C. With no additions to the medium, the thallus pH was 0.1-0.2 units lower than the surrounding water (Table 2-6). During the course of the measurements, the water pH dropped 0.1 unit in the light. In the presence of 40 mM KI, the thallus surface was up to 1.4 pH units lower than the surrounding water. Apparently, the presence of the KI stimulated H+ extrusion; the water pH dropped 0.4 units in the same



















15





10
o

0


5 O
E



0 I I

0 1 2 3 4 mM HCO3Figure 2-3. Effect of 40 mM KI on photosynthetic rate of Udotea thalli at pH 8.0. (0) control,
(0) 40 mM KI added.
Ln



















20











Eo

15




0
0 1 2 3 4 mM HCO,Figure 2-4. Effect of DIDS on photosynthetic rate of Udotea thalli at pH 8.0. (0) control,
(0) 0.24 mM DIDS added.





















30





-C
20 L
01



10








0 5 30 60 90 120 Incubation Period (min)



Figure 2-5. Effect of vanadate on photosynthesis of Udotea thalli. The vanadate solutions were prepared according to three different methods, as indicated in Materials and Methods. The pH of each reaction mixture with the vanadate solutions A, B, and C added, respectively, was different:
7.93 (0), 7.86 (jj), or 7.98 (0).

U,
or










56







Table 2-6. Surface pH measurements of Udotea thalli under 300 pmol m-2 s-1 irradiation (400-700 nm), except as noted, at 250C. The incubation medium for each was artificial seawater with 2 mM DIC added. The two pH values reported for the water were at the beginning and end of the experiment. Each set of values reported was for eight to ten measurements on one piece of thallus. The tip values were recorded at the natural apical end of the thalli.

Conditions Water Thallus Tip Thallus Center

pH
No additions 8.73 8.32 0.06 8.21 0.08 8.61 8.47 0.06 8.58 0.10 8.47 0.09 8.55 0.06
+ 40 mM KI 8.59 8.34 0.09 7.18 0.68 8.22 7.42 0.16 6.99 0.30
+ 0.24 mM DIDS 8.79 8.05 0.83 8.31 0.08 8.64 8.27 0.13 8.29 0.10
+ 1 mM Vanadate 7.54 7.61 0.14 7.44 0.03 7.48 7.23 0.12 7.42 0.02
No plant 8.90
8.77
Dark 8.80 8.43 0.09 8.39 0.09 8.84 8.41 0.19 8.42 0.04
Dark, No plant 8.92 --8.90









57

period. In the presence of 0.24 mM DIDS, the thallus surface was 0.40.6 pH units lower than the water, and the water also dropped 0.2 units during the readings. The presence of 1 mM vanadate lowered the water pH; however, the pH of the water did not change during the course of the measurements, and the thallus pH remained similar to that of the water. When no thallus was present in the water in the light, the pH dropped by about 0.1 unit over a 15 min period. In the dark, without a thallus piece, the water pH remained essentially unchanged. When a piece of thallus was incubated and measured in the dark, the pH of the water remained about the same, while the pH of the thallus was 0.4 pH units lower than the surrounding water, thus indicating that the reduced pH of the Udotea thallus surface, perhaps caused by H+ extrusion, was not dependent on light or photosynthesis.

The effect of 02 on photosynthesis at pH 8.2 was examined for the Udotea thalli (Table 2-7). The photosynthetic rate was inhibited 35 to 40% by 21% 02 as compared to 1% 02 in the gas phase. However, increasing the DIC had no effect on the degree of 02 inhibition of photosynthesis, thus suggesting that the 02 inhibition was not a photorespiratory phenomenon.

Discussion

The photosynthetic studies of Udotea thalli indicated that

although CO2 was used more readily for photosynthesis, there appeared to be some capacity for using HC03-. The CO2 could enter Udotea siphons by diffusing across the plasma membrane or it could be transported across, as hypothesized for the marine macroalga Stichococcus bacillaris (102), the green microalgae Chlorella ellipsoidea (127) and C. saccharophila (126), and the cyanobacterium Synechococcus sp. (7, 52). The CO2 pump in the colonial green alga Scenedesmus obliquus, grown on high CO2 and then adapted to air levels of C02, is accompanied by plasma membrane CA formation (143). Other species in the Chlorophyta, such as Ulva lactuca and U. fasciata, appear to take up HCO3- directly (6, 94), possibly by a










58







Table 2-7. Effect of oxygen concentration on photosynthesis in Udotea at pH
8.2 and 300 pmol quanta m- s- Values reported are the mean of 4 replicates SD.

DIC CO2 HCO3- 02 Photosynthetic 0 Rate Inhibition mM pM mM % pmol O g-1 FW h

1 9.84 0.93 21 4.85 1.62 --2 7.80 4.19 38
2 19.68 1.85 21 6.23 0.55 --2 10.43 1.42 40 5 49.20 4.64 21 10.40 2.02 --2 16.11 4.22 35









59

HC03-/OH- antiport system (12). In the green unicell Chlamydomonas reinhardtii, cells adapted to air levels of CO2 after growth at 5% C02 reportedly utilize HC03- in a DIC concentrating mechanism (39, 103). This mechanism also includes an appearance of external CA activity. Thus, CA may function as a component of either CO02 or HC03- utilization systems.

Carbon dioxide use in Udotea would have to be by diffusion or a CO2 pump that does not require external CA activity, as external CA activity was not measurable on Udotea thalli. Bicarbonate use in two of the Ulva species is hypothesized to occur by a HC03-/OH- antiport, but the pH of the Udotea thalli did not support the occurrence of OHextrusion. External CA activity is also a component of some HCO3utilization mechanisms, but this activity was lacking in Udotea. Thus, DIC utilization in this alga probably does not occur via any of these mechanisms.

The experiment modified from Cooper et al. (44) of investigating which species of DIC was used for photosynthesis indicated that CO2 use resulted in the most rapid photosynthetic rate. This experiment was predicated on a lack of external CA on the Udotea thallus. The experiment to determine the inorganic carbon substrate for photosynthesis was performed at pH 8.2 at 90C to slow the equilibration of the various carbon species. The reduced temperature also slowed the photosynthetic rate. When the inorganic carbon was added as largely HC03-, even adding exogenous CA to the system did not result in a rapid enough production of CO02 to raise the photosynthetic rate above that achieved when HC03- alone was present. Carbonic anhydrase facilitates the rapid establishment of the equilibrium among the various carbon species for a given pH. When most of the DIC was added as CO02, adding CA resulted in a rapid shift of most of the inorganic carbon to HC03-, the species that predominates at pH 8.2, and this reduced the photosynthetic rate substantially. Thus, it appeared that the









60

photosynthetic rate dependended on the low CO2 concentration, as Fig. 22 indicated that 0.03 mM CO02 could support the same rate observed in this experiment. If little or no HC03- use occurred, then the low concentration of CO02 present in seawater in equilibrium with air (10 pM) is a major factor for Udotea photosynthesis in the environment. The alga must have a mechanism other than HC03- utilization to elevate the internal carbon concentration above that in the surrounding water.

Although in a number of species of red, brown, and green microand macroalgae, external CA activity has been found (15, 63, 138, 141), Giordano and Maberly (61), in a survey of 34 species of macroalgae, were unable to find any correlation between the presence of CA activity and the relative ability to use HCO3- for photosynthesis, indicating that external CA activity is not always essential for HC03- utilization. However, CA activity does play a role in some HC03- use mechanisms, including that in the red alga Gracilaria tenuistipitata (63), where the external CA facilitates the conversion of HC03- in the medium to C02 which then diffuses across the plasma membrane. Conflicting reports have appeared for the red alga Chondrus crispus. Smith and Bidwell (138) reported the presence of external CA, while Giordano and Maberly

(61) did not find it to be present. The brown alga Ascophyllum nodosum uses HC03- and lacks external CA activity (78). In contrast, external CA activity was found in some brown algae of the Fucales and Laminariales (141). A difference exists with regard to the presence of CA in the green macroalgal genus Ulva. Bjirk et al. (15) report the presence of CA in U. rigida as do Giordano and Maberly (61) for U. lactuca, although the activity is low. In contrast, Drechsler and Beer indicate they found no external CA activity for U. lactuca (47).

No external carbonic anhydrase activity could be detected on the Udotea thalli by direct measurement. Thus, it is unlikely that a DIC utilization mechanism, if present, would have external CA activity as a component. Reiskind et al. (123) measured high levels of internal CA









61

activity in Udotea, not associated with the membranes, and thus presumably soluble. Their data suggest that CA is located in the chloroplasts, but the concurrent activity in the cytosol cannot be excluded. Activity of CA in the cytosolic compartment could play a role in maintaining a sufficient CO02 supply for the PEPCK enzyme that appears to be located in the cytosol (122,123) if HC03- be transported across the plasma membrane into the cytosol.

The final pH values attained in a pH drift experiment were 8.3 and 8.95, which are in the range of CO2-only users reported by Maberly (94). A low pH compensation point, the pH at which no net carbon exchange occurs, has been observed for sublittoral algae within each division, and this may be related to the growth conditions of these algae (6). Similarly, the low pH compensation point observed for Udotea as compared to the other algae examined by Maberly (94) may be related to the sublittoral growth conditions of Udotea. Maberly (94) indicates that the ability of a species to raise the pH and deplete the total available inorganic carbon varies with the habitat where the species grows. A major contrast exists between the macroalgae that grow in rockpools, which are very effective at removing inorganic carbon and those that are found subtidally in relatively low light, most of which have a more limited ability to use HC03-.

The final CO02 concentration attained by Udotea conglutinata of 5 pM was 25-fold higher that previously measured CO02 compensation points of 7 pL CO02 L-1 (0.2 pM) for Udotea flabellum (123), which also exhibits C4-like photosynthetic features. The pH drift experiment began with 2 mM equivalent to 0.12 mmol DIC present in the incubation bottles. Based on a HC03- dehydration rate of 0.29 pmol min-i, a total of 0.104 mmol C02 would have been produced in 6 h in the 60 mL incubation bottles, and assuming all the CO02 produced was used for photosynthesis, 0.016 mmol DIC would have remained at the end of that time period. However, the DIC measurements at the end of the incubation indicated that 1000 pM or









62

0.06 mmol DIC remained in solution. Thus, as the total DIC in the bottle became too low for the alga to photosynthesize, some of the carbon from its CaCO3 skeleton may have been utilized as a source of carbon to be reduced, perhaps avoiding potentially lethal photoinhibition.

At the end of the incubation period, 2 of the 3 replicates showed an increase in the alkalinity of the artificial seawater, from 1.36 at the start to 1.93 when a final pH of 8.21 was attained, and 2.58 when the final pH reached 7.89. This phenomenon has also been reported for other macroalgae in pH drift experiments, including Dilsea carnosa, Polysiphonia nigrescens, and Ceramium rubrum, all red algae (6). These species grow sublittorally, as does Udotea. It has been suggested that an increase in the alkalinity of the seawater was responsible for the excretion of inorganic carbon into the medium by the red algae (6). Such an increase in alkalinity of the water surrounding the thallus could have been caused by the reduction of an internal pool of nitrate (6, 70).

Photosynthetic rates for Udotea at pH values of 7.7, 8.2, and 9.0 demonstrated "classic" HC03- utilization by the alga (42, 77). Photosynthetic rates in solutions of different pH with the same concentrations of free CO2 and varying amounts of HC03- and CO32- were plotted versus CO2 concentration. As C032- is not used by plants for photosynthesis (93), the increasing concentration of HC03- relative to CO2 with pH was important. For a given DIC concentration, the proportion of the total DIC present as CO2 decreases with increasing pH. However, at the higher pH values, the photosynthetic rates were higher than measured at a lower pH for the same DIC concentration, thereby strongly suggesting that the HCO3- present was contributing to the increased photosynthetic rate observed.

Even though there was evidence for some HCO3- use in Udotea, the question must still be answered whether the ability of the alga to use









63
HC03- in addition to CO2 from the surrounding seawater is able to explain the observed C4-like photosynthetic features. The crucial part of this question is whether the alga is able to concentrate internal inorganic carbon above the level of the ambient solution so as to be able to shift the reaction of Rubisco to favor carboxylation and the photosynthetic carbon reduction cycle over oxygenation and photorespiration. To concentrate inorganic carbon internally requires the expenditure of energy (40) due to the thermodynamic constraints of the system. Photosynthesis in species that can use HC03- is substantially more than half-saturated with air-equilibrium DIC concentration, i.e., 10 MM CO2 and 2 mM HC03-, while those restricted to using CO02 are saturated to a lesser degree at the same DIC concentrations (94). Thus, the degree of saturation for the photosynthetic rate at ambient DIC concentration can be used as a guide to indicate whether a carbon concentrating mechanism is operational. In the red alga Gracilaria conferta, photosynthesis saturates at a DIC concentration close to ambient and exhibits 02-insensitive photosynthesis, thus suggesting the operation of some sort of carbon concentrating mechanism, perhaps HC03- utilization (73). Although internal DIC concentrations were not measured directly, the lack of saturation of photosynthesis in Udotea at ambient DIC concentration suggested that the alga was using primarily CO02 for photosynthesis.

The data obtained from the effect of 40 mM KI on photosynthesis provided more clues as to whether Udotea can use HC03-. It is possible that the K+ ion interfered with a Na+/HC03- symporter in the plasma membrane, the same sort of transporter reported to be operating in the marine diatom Phaeodactylum tricornutum (46). Potassium iodide did not exhibit an inhibitory effect until the concentration was increased to 40 mM, but the same concentration also was used in Ulva lactuca to inhibit photosynthesis at pH 8.2 (47). This is a high concentration, but if it was competing with Na+, the concentration of Na+ in the bathing medium









64
was 450 mM, so potentially an inhibitory effect occurred at one-tenth the concentration of the important ion. Another possibility is that the I- ion was in competition with HC03- for transport on a membrane translocator. This is an unlikely scenario, as the C1- concentration was greater than 450 mM, so it would be more likely to interfere with HC03- than I-. Also, HC03 was not able to reduce the inhibition. It cannot be ruled out that the I- concentration inside the siphons may have interfered with various cellular processes. In the presence of 40 mM KI, the pH of the thallus surface was 1.0-1.5 pH units lower than the control, where thalli were immersed in 2 mM DIC in unbuffered artificial seawater, indicating that the KI increased H+ extrusion or OH- uptake by the alga.

The pH microelectrode used for these measurements was placed in contact with the thallus, but Udotea conglutinata has a sheath external to the plant cell wall, and the role and chemical composition of this sheath are unknown (24). Thus, the pH electrode could not be placed immediately next to the cell wall. Nonetheless, the pH data indicated that the thallus was performing either H+ extrusion or OH- uptake at the surface. This acidification did not appear to be localized to particular regions, as the lower pH was found all over the whole thallus and on both sides. Polarity with respect to pH, as exhibited by the submerged aquatic angiosperm Potamogeton lucens on opposite sides of the leaves (139), or the green alga Chara in different regions of the cell wall (155) could not be detected. One caveat is that the size of the microelectrode tip (1.5 mm diameter) would preclude the detection of acid/alkaline banding patterns that were smaller than about 1 mm in width. In contrast to these Udotea data, measurements of surface pH of Ulva have indicated that this thallus has a much higher pH of 10, but it is also uniform with no detectable zones of acidification or alkalinization (12). Clearly, Ulva is not extruding H+.









65

The contrast in thallus pH measurements between Udotea and Ulva suggest a difference in the DIC utilization mechanisms. The H+ extrusion at the Udotea surface could cause HCO3- in the boundary layer to be converted to CO02, which could then either diffuse into the cytosol or be actively transported across the plasma membrane into the cytoplasm. Ulva, in contrast, is likely to be transporting HC03- across the plasma membrane into the cytosol via a HCO3-/OH- antiport, which would be accompanied by an increase in pH on the outside of the thallus

(12).

Calcification (100) or a Na+/H+ antiport system, as reported for Dunaliella salina (82) could be causing the H+ release However, when a section of Udotea thallus was incubated in the dark for 15 min, the plant surface was 0.4 pH units lower than the seawater in the incubation medium. Thus, photosynthesis or even the presence of light was not essential for the putative extrusion of H+. The pH change at the algal surface caused the artificial seawater pH to decrease by 0.4 units during the time the thallus pH was being measured. In the case where no plant was present and the artificial seawater was incubated in the light, the water pH dropped by 0.1 unit over a 15-min period, probably due to a temperature increase in the water, so most of this acidification of the external water could be attributed to the presence of the macroalga.

The inhibition of photosynthesis in Udotea that occurred in the presence of DIDS suggested the presence of an anion exchanger in the plasma membrane. In red blood cells, a protein located in the plasma membrane called the band 3 anion transporter (anion exchanger) facilitates HC03- transport, but this is not an active transport system, and thus does not require expenditure of ATP energy (53, 135). In red blood cells, this protein catalyzes the transmembrane exchange of one HC03- for one Cl-. The inhibitor DIDS, used in this study of Udotea, competes quite specifically with Cl- for binding at a lysine residue in









66

the vicinity of the outward-facing transport site and inhibits inwardfacing sites by shifting their conformation to that of the outwardfacing site (53). This band 3 anion transporter is also located in the plasma membrane of Ulva, as DIDS was able to reduce the photosynthetic rate when HC03- but not CO2 was available as the external DIC species to be taken up by the alga (47). In the presence of DIDS, the surface pH of the alga was 0.2 to 0.3 units more acidic as compared to the control, although H+ translocation is not reported as a function of the anion exchanger.

Based on data from thallus incubation with vanadate, ATP may be necessary for utilization of HC03-, as after 2 h incubation in 5 mM Na3VO4, photosynthesis was inhibited 34%. For intact cells of Dunaliella parva, where a plasma membrane ATPase plays a role in photosynthesis, 77% of the ATPase activity was inhibited after 3 h incubation in vanadate (60). For intact Scenedesmus and Chlamydomonas cells, vanadate inhibited plasma membrane ATPase activity after 90 min incubation (114). In the presence of vanadate, Udotea thallus surface pH remained unchanged over time, and the pH of the water did not decrease, suggesting that the putative H+ extrusion was eliminated. These data may lend further support for the presence of a plasma membrane H+ translocating ATPase. If the ATP synthesis is halted, no proton movement occurs, as the two processes are intimately tied together, according to the chemiosmotic hypothesis. Thus, if a HC03translocator is operating in Udotea, it appears to require ATP for the transport and so be able to accumulate DIC inside the cell at a concentration higher than that found in the surrounding medium. However, after the 2 h incubation with vanadate, photosynthesis was decreased only 34%, thus suggesting that other DIC utilization mechanisms were operating to maintain the photosynthetic rate.

A comparison was made between the measured rate of photosynthesis and that predicted if photosynthesis was limited by the rate of CO2









67

production from the spontaneous dehydration of HC03-. This analysis is complicated by the process of calcification that occurs in udotea conglutinata. In Chara, it has been demonstrated that photosynthesis and calcification occur in about a 1:1 ratio (99). Using 02 evolution as a measure of carbon metabolism in photosynthesis assumes that for every mole of inorganic carbon fixed, one mole of 02 is evolved. But when considering the effect of calcification in this picture, it must be considered that when the alga is photosynthesizing, each photosynthesis "event" requires the uptake of 1.25 moles of inorganic carbon: one is fixed by Rubisco in the chloroplast and after four fixations, precipitation occurs at the surface with Ca2+ to form carbonate (23). Inorganic carbon use in Udotea in light of these two processes must then be considered. At 2 mM DIC and pH 8.2, the ambient conditions found in natural seawater, 0.58 pmol of CO2 was produced per hour in the closed system of the 02 electrode chamber based on the kinetics of HC03dehydration. In 21% 02, the photosynthetic rate was 0.44 pmol h-1, indicating that CO2 was supplied at a rate that exceeded the uptake rate. If calcification is considered in addition to the measured 02 evolution, then the carbon really used by the alga for photosynthesis was 0.55 pmol h-1. The CO2 supply rate of 0.58 pmol h-1 still met the inorganic carbon requirements of the algae. At 2 mM DIC and 2% 02, the photosynthetic rate was 0.78 pmol h-1. Under these conditions, CO2 could not be produced fast enough to meet the photosynthetic requirement, so HCO3- use may have made up the deficit. In 1 mM DIC with a HC03- dehydration rate of 0.29 pmol h-1, under both 21% and 2% 02, HC03- would have to be used to meet the requirement for photosynthetic carbon and then additional HC03- used for calcification. When the alga was placed in 5 mM DIC, more than twice the concentration the plant is exposed to in nature, C002 was supplied from HCO3- dehydration at 1.46 pmol h-1, a rate higher than the 02 evolution in both 21% and 2% 02, thus indicating that the C02 supply was sufficient for both carbon fixation









68

and calcification. Thus, it appears that Udotea does have some capacity for HCO3- utilization above and beyond the use of free CO2.

Although 02 inhibited the photosynthesis of Udotea, it was unlike the phenomenon observed in C3 plants. The photosynthetic characteristics of Udotea flabellum clearly indicate that its metabolism is C4-like in nature (123), and some earlier work on U. conglutinata indicated the same metabolism as the other species (Reiskind & Bowes, unpublished). The apparent 02 sensitivity of photosynthesis may be attributed to other 02 effects, such as the Mehler reaction, mitochondrial respiration, or the glycolate pathway, similar to that found in the red alga Chondrus crispus, where 02 inhibition is relatively insensitive to CO2 concentration and not completely reversed by high CO2 (30).

Because Udotea conglutinata is a calcareous alga, the skeleton of carbonates plays a role in photosynthesis in addition to providing structural integrity for the intertwined siphons of the thallus. In species that calcify, the process does not occur until the cells are photosynthetically competent (24). In the presence of CaC12 at 2 mM DIC and 21% 02, twice as much DIC was removed from the medium as 02 was evolved. This proportion suggested that 2 moles of inorganic carbon were required to complete the whole photosynthetic process in Udotea of 02 evolution and calcification. At 5 mM DIC, nearly 10 times more DIC was removed from the medium as 02 was evolved, but the quantity of 02 evolved was approximately equal to that in 2 mM DIC. A portion of this inorganic carbon could be used in calcification, but the reason for this difference is not known at this time. When CaC12 was withheld from the photosynthetic medium, photosynthesis was measurable as 02 evolution in the 02 electrode, but the amount of inorganic carbon in the medium actually increased during the course of the 15 min measuring period. Thus, it may be that the plant was excreting inorganic carbon at its









69

surface which normally would complex with Ca2+ to form CaCO3 in the skeleton. Chara also does not calcify under low Ca2+ conditions (99).

Calcification in Udotea appears to occur by a mechanism similar to that in Halimeda, a closely related siphonaceous species from the same family (23). Calcium carbonate precipitates as aragonite crystals in the intercellular spaces which are separated from the external seawater. In Halimeda, swollen ends of the siphons, called utricles, cause this isolation and the sheath found outside the cell wall in Udotea conglutinata may serve a similar function. The model for Halimeda states that about 70% of the cell wall area faces these intercellular spaces. When CO2 uptake occurs from the carbon in the intercellular spaces, the pH increases as does the concentration of CO32- in these secluded spaces and Ca2+ from the surrounding medium moves through the cell walls to these areas, thereby facilitating CaCO3 precipitation

(23). The photosynthetic removal of carbon is greater than the resupply rate from the external seawater and cellular respiratory processes. The surface acidification in the presence of CaCl2 in the seawater is a byproduct of the precipitation of CaCO3 (100).

Studies in Chara also have provided important information about

the calcification process. Protons generated from the hydroxylation and precipitation of CO2 allow the plant to manufacture twice the amount of CO2 used in calcification (99). The model for Chara suggests that the inorganic carbon and Ca2+ for the process come from inside a heavily encrusted plant, and a Ca2+/ATPase appears to be involved in part of the transport (99). The H+ must be removed from the calcification site; perhaps H+ are transported by cytoplasmic streaming and through a large, acidic vacuole to another part of the plant cell. In fact, H+ generation may be the principal benefit of calcification (99). This precipitation occurs at an appreciable rate in nature, as 50% of the dry weight of the Udotea thalli was composed of carbonate salts, and a similar proportion occurs in Characeans (100).









70

Calcification in Udotea could occur in the intercellular spaces, which are really spaces between the siphons. As CaCO3 precipitation occurred, the H+ generated were translocated to the outer surface of the thallus, and this H+ extrusion was recorded in this investigation. The calcification process was actually quite similar to that for Chara, with H movement occurring across the same cell, or siphon in this case, except that only the acidification could be measured because the removal of H+ occurred inside the thallus. However, in Udotea, some H+ extrusion continued in the absence of photosynthesis.

The current hypothesis regarding inorganic carbon utilization in Udotea is that the alga preferentially uses CO02 when it is available, but limited HC03- use also occurs. The HC03- is probably transported by a carrier protein in the plasma membrane in a facilitated manner, such that no energy expenditure is required, and thus internal accumulation of DIC does not occur above the external concentration. However, it also appears as if some ATPase is involved, but no external CA. ATPase may be involved in calcification or conversion of HC03- to CO02 by acidification and thus H+ extrusion.

Thus, the model for photosynthesis in Udotea includes DIC uptake in the form of C02, which is produced in the boundary layer by H+ extrusion associated with the calcification process. This uptake could occur via diffusion or active transport of the CO02 across the plasmamembrane. Some HC03- uptake across the plasmamembrane also occurs probably on an anion exchanger that operates via facilitated transport. As inhibition of these mechanisms inhibited photosynthesis by no more than 34%, the high phosphoenolpyruvate carboxykinase activity, reportedly located in the cytosol, is the key component of the photosynthetic mechanism in Udotea to concentrate inorganic carbon for Rubisco and thus, cause the macroalga to exhibit its C4-like features.

















CHAPTER 3
ROLE OF PHOSPHOENOLPYRUVATE CARBOXYKINASE IN THE
PHOTOSYNTHESIS OF THE MARINE MACROALGA UDOTEA Introduction

Udotea is a green, marine, macroalga which exhibits C4-like photosynthetic characteristics, despite lacking the Kranz anatomy associated with terrestrial C4 species. The thallus of this alga is composed of intertwined coenocytic siphons. Although a "primitive" plant, this alga appears to utilize a more "advanced" type of photosynthetic metabolism, initiated by the enzyme PEPCK, as when PEPCK is inhibited by MPA, the photosynthetic metabolism of this alga appears C3-like (122). The focus of this investigation was to examine the kinetics of PEPCK in order to ascertain how this enzyme could be functioning as a component of the C4-like metabolism. In some higher plants that utilize C4 photosynthesis, PEPCK functions as a decarboxylase in the cytosol of bundle sheath cells (4). In the brown algae, PEPCK is hypothesized to function in an anapleurotic role to supply the cells with carbon skeletons for the synthesis of other compounds (159). PEPCK is also found in a variety of other organisms ranging from bacteria and yeast to diatoms and vertebrates. In the plants and diatoms, PEPCK uses ADP/ATP, while the enzyme in Euglena and vertebrates utilizes GDP/GTP for its activity (117, 152). In mammals, PEPCK is a critical enzyme in gluconeogenesis, involved in removing CO2 from OAA to produce PEP for the eventual production of glucose.

Materials and Methods

Materials

The marine macroalga Udotea flabellum (Ellis and Solander)

Lamouroux (division Chlorophyta) was collected during the summer on the north side of Marathon Key, Florida, in Florida Bay in water ranging 71









72

from 0.5-2.5 m in depth. After collection, the plants were kept outdoors for up to two days in a fresh seawater-fed and aerated tank until frozen in liquid nitrogen (LN2). Enzyme Extraction

For experiments on crude extract, 1-2 g of LN2-frozen plant

material was ground in a mortar and pestle with sand at 40C in 3-6 mL of 50 mM Hepes-NaOH, 2 mM MnC12, 5 mM DTT, 1% (w/v) PVP-40, 1 mM PMSF, and 10 pM leupeptin at pH 7.0. The homogenate was centrifuged at 13,000 g for 2 min in a microfuge at 400C. The supernatant was then desalted through either a 5 mL Sephadex G-25 or BioRad P-6 column with the same medium used for grinding, but without PVP-40. The column eluant collected just after the void volume was then used for subsequent enzyme assays.

For isolation of Udotea PEPCK, approximately 45 g of frozen plant material was powdered in LN2 and then ground with a mortar and pestle and sand at 40C in 150 mL of a medium containing 50 mM MES-NaOH, 5 mM MnC12, 5 mM MgC12, 5 mM DTT, 0.2 mM Na2EDTA, 1 mM PMSF, and 1% (w/v) PVP-40 at pH 6.5. Following centrifugation at 40C for 15 min at 12,000 g, the pellet was discarded, and the supernatant was used for protein isolation. Solid (NH4)2SO4 was added slowly, with constant stirring, to the protein solution kept on ice. The protein that precipitated between 40 and 50% (w/v) (NH4)2SO4 was pelleted at 3500 g and resuspended in 6 mL of the elution buffer, which consisted of 50 mM MES-NaOH, 5 mM MnC12,

5 mM MgCl2, 5 mM DTT, and 1 mM PMSF at pH 6.5. The solution was then brought to 20% (v/v) glycerol.

Enzyme Purification

The protein solution was loaded onto a Sephacryl S-300 column (78 x 2.5 cm, 380 mL bed volume) and eluted at 40C with the buffer described above. Fractions were analyzed for protein by measuring the absorbance at 280 nm in a spectrophotometer. Fractions were also analyzed for PEPCK, malate dehydrogenase (MDH), and Rubisco activities. Those









73

fractions containing high PEPCK activity and low MDH activity were loaded onto a Sepharose CL-6B column (25 x 1.5 cm, 40 mL bed volume) for ion exchange chromatography and eluted with a linear 0-0.3 M KCl gradient in the same buffer used for gel filtration. Fractions were analyzed for PEPCK, MDH, and Rubisco activities, and protein. The fractions containing highest PEPCK activity were pooled (total volume 35 mL), the protein concentration was determined, 2 mg mL-1 bovine serum albumin (BSA) was added, and the solution was frozen in LN2 for further enzyme studies.

Enzyme Assays

Enzymes were assayed at 2500C (except where noted). The carboxylation activity of PEPCK was routinely measured spectrophotometrically at 340 nm by the MDH/NADH-coupled reaction (151). The standard assay mixture consisted of 50 mM PIPES-NaOH, 5 mM MnC12, 30 mM NaHCO3, 5 mM DTT, 20 to 30 pL enzyme extract, 2 units MDH, 0.2 mM NADH, 3 mM ADP, and 10 mM PEP at pH 6.8 in a total volume of 0.5 mL. The reaction was initiated by adding ADP and followed for a period of 3 minutes during which the reaction proceeded at a linear rate. An absorption coefficient of 6.22 mM1 cm-1 was used for NADH. The PEPCK carboxylation activity was also measured radiochemically in a standard assay of 50 mM PIPES-NaOH, 5 mM MnC12, 30 mM NaH14CO3 (2 pCi pmol-1), 5 mM DTT, 20 to 30 pL enzyme extract, 2 units MDH, 0.2 mM NADH, 2 mM ADP, and 7 mM PEP at pH 6.8 in a total volume of 0.5 mL. The reaction was initiated with PEP and terminated after 6 min with 0.1 mL 6 N HC1 saturated with 0.13 M dinitrophenylhydrazine. After drying at room temperature to allow unreacted NaH14CO3 to escape, the sample was rehydrated with 0.4 mL deionized water, one drop of 6 N NaOH, and 4 mL liquid scintillation cocktail (RPI 3a70). The capped vials were then vortexed. The acid-stable products were counted in a liquid scintillation counter.









74

The decarboxylation activity of PEPCK was measured

spectrophotometrically at 340 nm by the MDH/NADH-coupled reaction modified from Burnell (33). The assay mixture consisted of 50 mM Bicine-NaOH, pH 7.8, 5 mM MnC12, 20 to 30 pL PEPCK enzyme extract, 0.25 mM NADH, 1 unit lactate dehydrogenase, 2 units pyruvate kinase, 0.5 mM ATP, and 0.5 mM OAA, at pH 7.8, also in a total volume of 0.5 mL. The reaction was initiated with OAA and followed for 3 min, during which time the reaction proceeded at a linear rate.

The activity of MDH was assayed spectrophotometrically at 340 nm by measuring NADH oxidation in a method modified from Yueh et al. (161). The assay mixture was composed of 50 mM Bicine-NaOH, 1 mM Na2EDTA, 0.2 mM NADH, 10 ML enzyme extract, and 0.5 mM OAA at pH 8.0. The reaction was initiated with OAA.

The activity of Rubisco was measured radiochemically in an assay mixture consisting of 50 mM Bicine-NaOH, 10 mM MgC12, 50 pL enzyme extract, 5 mM DTT, 5 mM isoascorbate, 10 mM NaH14CO3 (0.5 pCi pmol-1), and 1 mM RuBP at pH 8.0. The enzyme was activated for 5 min at 300C in the assay mixture without RuBP present (128). The reaction was initiated with RuBP, performed at 300C, and terminated after 30 s with 6 N HC1. After drying in a 700C oven and rehydrating the samples, the radioactivity of the acid-stable products were measured as described above.

Protein Assay

During elution of the columns, protein concentration was

determined by measuring the absorbance of the protein solution at 280 nm, but in all other instances, it was determined by the method of Bradford using bovine F-globulin as standard (29). Enzyme Storage Conditions

Following purification, aliquots of PEPCK were stored for a period of 5 weeks as enzyme solution in elution buffer, with and without 2 mg mL BSA, or as the precipitate of 65% (NH4)2SO4 in the refrigerator









75

(40C), freezer (-200C), or in LN2 (-1960C). The carboxylation activity of the aliquots was measured after 2 weeks and again after 5 weeks of storage.

Enzyme Kinetic Analyses

The inorganic carbon species utilized for carboxylation by Udotea PEPCK was determined by the method of Cooper et al. (44) at 120C so as to slow equilibration among the inorganic carbon species. The DIC was added at a concentration of 8 mM as either HCO3- or CO02 to initiate the reaction in the presence or absence of CA. To provide predominantly HC03- to the enzyme, a solution of 0.5 M NaHCO3 was added directly from a Hamilton syringe into the assay mixture in the cuvette. However, to provide predominantly CO02, NaHCO3 solution was drawn up into a Hamilton syringe followed by an equal volume of 0.5 N HC1. After dissolved C02 was produced in the syringe, the solution was dispensed into the cuvette where the reaction was to occur. The reactions were performed at pH

8.0. All solutions, except NaHCO3, were prepared C02-free by bubbling with N2. This bubbling was continued after the mixture components, except for the DIC, were combined in the cuvette, so that the assays were performed under C02-free conditions. The DIC was added last to initiate the reaction. The reaction was followed for only one minute in the spectrophotometer so that the measured rate would be dependent upon the inorganic carbon species initially added to the reaction mixture.

The affinity of PEPCK for the carboxylation substrates, DIC, ADP, and PEP and the decarboxylation substrates, OAA and ATP was examined. The effect of Mn2+ was investigated for both reactions. The concentration of the substrate in question was varied while the other reaction mixture components were held constant at saturating concentrations. The constants were calculated from Eadie-Hofstee plots. The computer program Enzfitter was also used to calculate MichaelisMenten constants.









76

The carboxylation reaction was performed in the presence of ADP, GDP, or IDP, and the decarboxylation reaction was run with ATP, GTP, or ITP to determine how these components affected the reaction.

The effects of 0.5 mM OAA and 0.5 mM ATP on the carboxylation reaction and 1 and 3 mM DIC, 0.02 mM ADP, and 4 mM PEP on the decarboxylation reaction were examined. For both carboxylation and decarboxylation, the effect of 100 pM MPA on Udotea PEPCK was investigated. The effects of the intracellular metabolites malate, aspartate, F6P, F-1,6-BP, 3-PGA, and dihydroxyacetone phosphate (DHAP) were examined for both reactions. These potential effectors were added at 4 mM over a range of subsaturating concentrations for one reaction component at a time. Other substrates were present in saturating concentrations.

The carboxylation reaction was run at subsaturating DIC

concentrations and the decarboxylation reaction at subsaturating OAA concentrations in 0, 21, and 100% gas phase 02, (0, 0.46, and 2.18 mM 02 in solution) to determine whether 02 was an effector of either the carboxylation or decarboxylation reactions.

Results

In initial experiments, assays of PEPCK activity in both the carboxylating (pH 6.8) and decarboxylating (pH 7.8) directions were performed with crude extracts of Udotea thalli subject only to gel filtration through a 5 mL column of Sephadex G-25. The Vmax values from the preparations ranged from 0.69 to 1.32 pmol NADH mg-1 protein min-1 and 0.51 to 0.98 pmol NADH mg-1 Chl min-i for the carboxylation reaction, but were approximately four- to five-fold lower for the decarboxylation reaction, ranging from 0.14 to 0.34 pmol NADH mg-1 protein min-i and 0.10 to 0.25 pmol NADH mg-1 Chl min-1. The Km values for the various substrates are listed in Table 3-1. In the carboxylating direction, the enzyme exhibited an almost 100-fold concentration difference in Km values, with the lowest Km being for ADP and the highest for dissolved










77















Table 3-1. KM values for PEPCK activity in crude extracts of Udotea thalli. The values were derived from Eadie-Hofstee plots.

Substrate Km


mM

Carboxylating Direction

ADP 0.075 PEP 0.920 DIC 7.326

Decarboxylating Direction

ATP 0.023 OAA 0.028










78

inorganic carbon. In contrast, the affinities of the substrates ATP and OAA for decarboxylation were similar and comparable to the low Km(ADP) for carboxylation.

In an experiment where the same concentration (5 mM) of GDP or IDP was substituted for ADP, carboxylation proceeded at less than 20% of the ADP-catalyzed rate (Table 3-2). Similarly, for the decarboxylation reaction, the rate with 0.4 mM GTP or ITP was considerably reduced when compared to the rate with the same concentration of ATP. The nucleotide preference of the decarboxylation reaction was less specific than that of the carboxylation reaction, as the decarboxylation rate proceeded more rapidly when other nucleotides were substituted for ATP.

An examination of the metal ion requirement for PEPCK in crude extracts indicated that Mn2+ was essential for maximum catalytic activity in the carboxylation direction (Fig. 3-1). When Mg2+ was supplied at the same concentration as Mn2+, catalysis was only 10 to 25% of the rate with Mn2+; thus, Mg2+ alone could not substitute for Mn2+. If both species were present at the same concentration, Mg2+ inhibited the rate obtained in the presence of only Mn2+ by 5% and 44% at 2 and 5 mM ion concentration, respectively.

Udotea PEPCK was purified in a three-step procedure involving (NH4)2SO4 precipitation, which removed a portion of the extraneous cellular proteins, followed by separation of the proteins of interest according to molecular weight using gel filtration, and finally an ionexchange column. Protein was measured by reading the absorbance at a wavelength of 280 nm, and the presence of PEPCK was detected by its carboxylation activity. Two other enzymes, MDH and Rubisco, were also detected by assay of their activity in various fractions. The gel filtration step separated PEPCK from most of the MDH, but some MDH activity remained (Fig. 3-2). This step also eliminated the remainder of the Rubisco activity associated with the PEPCK. For the next step in the purification procedure, only the PEPCK fractions preceding the first










79















Table 3-2. Effect of various nucleotides on crude PEPCK activity. The carboxylation rate was 0.642 0.018 pmol NADH mg-1 protein min with ADP and the decarboxylation rate was 0.299 0.021 pmol NADH mg-1 protein min with ATP.

Relative Reaction Rate

Substrate Carboxylation Decarboxylation



ADP 100 --GDP 17 --IDP 10 --ATP --- 100 GTP --- 38 ITP --- 55



















0.30






C
z 0.20











0.00
1 2 3 4 5 Ion Concentration (mM)




Figure 3-1. Effect of either Mn2+ (0) or Mg2+ (i) or both ions present together at equal concentrations
(I) on PEPCK carboxylation activity from crude extracts.
00
O




















3.00 1.20


2.50
S 0.90
N
8 2.00

O
1.50 O.60 C


1.00

E 0.30
0.50


0.00 0.00
0 60 120 180 240 300 360 420

Elution Volume (mL)




Figure 3-2. Elution profile of the proteins PEPCK (0), MDH (0), and Rubisco (A) according to molecular weight from the Sephacryl S-300 column. Protein concentration (0) was measured at wavelength 280 nm as the fractions eluted from the column.









82

major MDH peak (i.e., elution volume 130 through 210 mL) were selected. These fractions with high PEPCK activity were pooled and loaded onto the ion exchange column. After rinsing with buffer lacking KC1, elution was accomplished with a 0-0.3 M linear KC1 gradient in the elution buffer. Ion exchange reduced the proportion of MDH in the PEPCK fraction such that the pool of the most active PEPCK had an average activity ratio of 7 units PEPCK to one unit of MDH (Fig. 3-3). This step also separated the remaining Rubisco activity from the PEPCK. After gel filtration and ion exchange chromatography, PEPCK was purified 12.6-fold, resulting in a specific activity of 18.52 pmol NADH mg-1 protein min-1 (Table 3-3).

To test its stability and determine optimum storage conditions, the isolated enzyme was stored under a variety of conditions for up to five weeks. Three temperatures were utilized (4, -20 and -1960C) and the enzyme was maintained in the ion exchange elution buffer or with 2 mg mL-1 BSA, or as precipitate in 65% (w/v) (NH4)2SO4 (Table 3-4). After

2 weeks, the enzyme stored as a solution in buffer or as a precipitate with (NH4)2SO4 at 4 and -20 OC lost nearly all activity, whereas that kept in LN2 at -1960C still retained about one-third of the initial activity. The enzyme stored with BSA retained between 70-90% of its initial activity at all the temperatures. After 5 weeks, the enzyme stored with BSA at -200C had lost half of its original activity, while the samples stored at 4 and -1960C retained 66% of the initial activity. Based on these experiments, the purified enzyme was routinely stored at -1960C after adding 2 mg mL-1 BSA to the solution, and this solution was utilized to characterize the purified enzyme.

When measured as a function of pH, the carboxylating and

decarboxylating activities of isolated PEPCK exhibited peaks of activity ranging from 6.0 to 7.5 and 7.5 to 8.0, respectively (Fig. 3-4). At pH

7.6, the rates of the two activities were equivalent, but at its optimum pH of 6.8, the carboxylation rate was 136% of the decarboxylation rate at its optimum of pH 7.8.



















1.50 0.80



1.20
>I- 0.60
N

0.90
E 0.40

0.60 o


E 0.20 't 0.30



0.00 -- 0.00
0 40 80 120 160 200 Elution Volume (mL)




Figure 3-3. Elution profile of the proteins PEPCK (0), MDH (0o), and Rubisco (A) according to charges on the molecules from the Sepharose CL-6B column. Proteins were eluted with a 0 to 0.3 M KCl gradient
(0). Protein concentration (0) was measured at wavelength 280 nm as the fractions eluted from the column., W)



















Table 3-3. Isolation of PEPCK from Udotea.

Purification Volume Protein Enzyme Specific Recovery Purification
Step Units Activity

mL mg pmol min-1 units mg-1 % -fold


Crude 162 57.51 84.54 1.47 100



40-50%
(NH, )SO 6 --- 148.36 --pellet,
resuspended



Sephacryl 74 23.75 12.43 0.52 15 0.35 S-300



Sepharose 38 0.46 8.52 18.52 10 12.6 CL-6B









85






Table 3-4. Effect of storage conditions on purified PEPCK carboxylation activity. The initial activity was 15.17 pmol NADH mg-1 pro min-.

Storage Conditions

Temperature Time Addition Activity Remaining
OC Weeks pmol NADH mg-1 pro min-i % 4 2 Buffer 0.42 3 + BSA 13.83 91 +(NH4)2SO4 0.67 4 4 5 Buffer 0.12 1 + BSA 10.00 66 + (NH4)2SO4 0.32 2

-20 2 Buffer 1.69 11 + BSA 10.42 69 + (NH4)2SO4 0.77 5
-20 5 Buffer 0.60 4 + BSA 7.68 51 + (NH4)2SO4 1.43 9

-196 2 Buffer 4.88 32 + BSA 13.42 88 + (NH4)2SO4 4.21 28

-196 5 Buffer 1.84 12 + BSA 9.86 65 + (NH4)2SO4 3.37 22




















40


c E 30
0
C. 0 O


E 20 O F 20
T



i 10





5 6 7 8 9 10 pH




Figure 3-4. Effect of pH on the carboxylation (0) and decarboxylation (0) activities of purified PEPCK at 250C.









87

The carboxylating and decarboxylating reactions of PEPCK exhibited a similar optimum temperature of 350C when measured at their respective pH optima (Fig. 3-5). The carboxylation reaction for the purified enzyme was measured as a function of DIC concentration at the optimum pH for carboxylation, 6.8, and at the optimum pH for decarboxylation, 7.8 (Fig. 3-6). The curve at pH 7.8 is flatter than that at 6.8; however, at both pH values, the reaction was saturated at 30 mM DIC with a K0.5(DIC) of 7.8 mM at pH 6.8 and 15.5 mM at pH 7.8. In solution, DIC exists as free CO2, H2CO3, HC03-, and C032- with the relative proportions dependent upon the pH and temperature of the solution. The relative species proportions were calculated from the Henderson-Hasselbach equation. The pKal of 6.36 for carbonic acid (H2CO3) in freshwater at 250C was used for the calculations (146). At pH 6.8, 0.273 of total DIC is CO2 and 0.727 is HCO3-, and at pH 7.8, 0.035 of total DIC is CO2 and

0.965 is HCO3-. At these pH values, the proportion of CO32- was negligible. Using these proportions, it was possible to calculate the K0.5 values for CO02 and HCO3- at the two pH values used in the assay. At pH 6.8, using a Michaelis-Menten plot (Enzfitter program), the K0.5(CO2) was calculated to be 2.1 mM and the K0.5(HCO3-) was 5.6 mM, while at pH 7.8 the K0.5(C02) was 0.5 mM and the K0.5(HC03- ) was 15.0 mM. Thus, the K.5(CO2) at pH 7.8 was four-fold lower than at pH 6.8, whereas the K0.5(HC03-) was 2.5-fold lower at pH 6.8.

Further investigation of the carboxylation reaction showed that it proceeded most rapidly when free CO2 rather than HCO3- was provided to the enzyme (Table 3-5). This assay was run at 12oC to reduce the rate of spontaneous equilibration between CO2 and HCO3-, and the reaction rates were lower as a result of the decreased temperature. Adding CA to speed the equilibration between CO02 and HC03- decreased the carboxylation rate by 36% when CO02 was the inorganic carbon source. When HCO3- was the predominant form of Ci supplied to the enzyme, the reaction rate was just 28% of that in the presence of C02. Adding CA in




Full Text

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INORGANIC CARBON UTILIZATION AND THE ROLE OF PHOSPHOENOLPYRUVATE CARBOXYKINASE IN THE PHOTOSYNTHESIS OF THE MARINE MACROALGA UDOTEA By BETH ANN BURCH A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 1993

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To My Dear Husband, Greg and In Loving Memory of My Parents, John and Doris Lilly

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ACKNOWLEDGMENTS I wish to thank Dr. George Bowes, my major professor, for his support and encouragement throughout my graduate career. The helpful suggestions provided by the supervisory committee, Dr. Steve Davis, Dr. Richard Smith, Dr. Charlie Guy, and Dr. Charles Allen are gratefully acknowledged. A special note of appreciation to Dr. Steve Davis, who so graciously volunteered to take his personal boat to Crystal Bay at all times of year in order to collect some of the plant samples. I appreciate the suggestions and help with research methodologies given by Dr. Julia Reiskind during the course of my tenure in Dr. Bowes' laboratory. A thank you goes to Dr. Charles Guy for allowing me to use some of his laboratory eguipment in experiments. I am grateful to the research staff of the laboratory of the Florida Keys Land and Sea Trust in Marathon, Florida for their help in collecting some of the plant samples and for use of the laboratory facilities. This research was funded by the United States Department of Agriculture, Science and Education Administration, Competitive Research Grants Office, Photosynthesis Program Grants 88-37130-3703 and 90-37130-5576. I am indebted to my husband, Greg, for his enduring love and support through my completion of this research and dissertation.

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TABLE OF CONTENTS ACKNOWLEDGMENTS LIST OF TABLES LIST OF FIGURES KEY TO ABBREVIATIONS ABSTRACT CHAPTERS 1 GENERAL INTRODUCTION AND LITERATURE REVIEW. The Marine Macroalga Udotea Habitat and Morphology Calcification Photosynthesis C 3 Photosynthetic Category C^ Photosynthetic Category CAM Photosynthetic Category Marine Algal Photosynthesis Chlorophyta Phaeophyta Rhodophyta Bacillariophyta Summary Phosphoenolpyruvate Carboxykinase Algae Higher Plants Fungi Yeast Anaerobe of rumens Bacteria Protozoa Platyhelminthes Nematodes Vertebrates Chickens Rats Other mammals 2 INORGANIC CARBON UTILIZATION IN THE MARINE MACROALGA UDOTEA Introduction Materials and Methods Materials Gas Exchange Experiments pH Drift Experiments Carbonate Salts in the Algal Skeleton. Page iii vi viii 1 1 2 3 3 5 9 10 12 16 20 22 22 23 24 25 28 28 28 29 29 30 30 31 31 33 33 36 36 37 37 37 40 41 IV

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Page Carbonic Anhydrase Assay 41 Thallus Surface pH 42 Chlorophyll Determination 42 Results 42 Discussion 57 3 ROLE OF PHOSPHOENOLPYRUVATE CARBOXYKINASE IN THE PHOTOSYNTHESIS OF THE MARINE MACROALGA UDOTEA 71 Introduction 71 Materials and Methods 71 Materials 71 Enzyme Extraction 72 Enzyme Purification 72 Enzyme Assays 73 Protein Assay 74 Enzyme Storage Conditions 74 Enzyme Kinetic Analyses 75 Results 76 Discussion 109 LIST OF REFERENCES 122 BIOGRAPHICAL SKETCH 135

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LIST OF TABLES Table Page 2-1 Proportion of Udotea dry weight composed of 43 carbonates. DW A refers to the dry weight remaining after acid treatment. 2-2 Effect of 10 mM CaClp on Udotea photosynthesis at 45 pH 8.2. Time elapsed during photosynthetic measurement was 15 min. Values reported are the mean of 2 measurements SD. 2-3 Final pH of three replicates of Udotea following a 47 pH drift experiment in 200 fjmol quanta rrf 2 s~ 1 for 6 hours at 32C. Values reported are the mean of 3 replicates SD. 2-4 Comparison of photosynthetic rates for Udotea 50 thallus sections measured at pH 8.2 and 300 pmol quanta rrf 2 s~ 1 and the predicted rates dependent upon the spontaneous rate of C0 2 production. The predicted rates were calculated according to Johnson (76). Measured photosynthetic rates are the mean of 4 replicates SD. 2-5 Measurement of hydration time for C0 2 at 10-12C, 51 recorded as a pH change of 2 units. Values reported were the mean of four or five replicates SD. The net photosynthetic rate at 2 mM DIC for the thalli examined for the presence of external CA was 26.47 5.77 pimol 2 g" 1 FW h" 1 2-6 Surface pH measurements of Udotea thalli under 300 56 fjmol rrf 2 s" 1 irradiation (400-700 nm) except as noted, at 25C. The incubation medium for each was artificial seawater with 2 mM DIC added. The two pH values reported for the water were at the beginning and end of the experiment. Each set of values reported was for eight to ten measurements on one piece of thallus. The tip values were recorded at the natural apical end of the thalli. 2-7 Effect of oxygen concentration on photosynthesis in 58 Udotea at pH 8.2 and 300 ^mol quanta nf 2 s~ 1 Values reported are the mean of 4 replicates SD 3-1 K values for PEPCK activity in crude extracts of 77 Udotea thalli. The values were derived from EadieHofstee plots. 3-2 Effect of various nucleotides on crude PEPCK 79 activity. The carboxylation rate was 0.642 0.018 /jmol NADH mg~ 1 protein min ~ 1 with ADP, and the decarboxylation rate was 0.299 0.021 ,umol NADH mg protein min _1 with ATP. vi

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3-3 Isolation of PEPCK from Udotea. 84 3-4 Effect of storage conditions on purified PEPCK 85 carboxylation activity. The initial activity was 15.17 ^mol NADH mg 1 pro min -1 3-5 Form of dissolved inorganic carbon utilized by 90 Udotea PEPCK for carboxylation. The reaction was performed at 12C to slow the equilibration between C0 2 and HC0 3 in solution. 3-6 K m va l ues for substrates of isolated PEPCK at 92 optimal pH for the reactions determined from EadieHofstee plots. The K m values for CC> 2 and HCO ~ at each pH were calculated based on the HendersonHasselbach equation using the pK al of 6.36 for carbonic acid in freshwater at 25 b C (146). 3-7 Effect of oxygen in the gas phase on the 93 carboxylation and decarboxylation activities of isolated PEPCK at various Die and OAA concentrations 3-8 Influence of metabolites on PEPCK reactions. All 97 reaction components were present at saturating concentrations for each reaction, except the effect of the metabolite on the reaction for a given substrate was measured at subsaturating concentrations of that particular substrate. Inhibition is reported vs. 1 mM substrate concentration for carboxylation and vs. 0.02 mM substrate for decarboxylation (unless noted differently) 3-9 Influence of various metabolites and MDH/NADH on 108 the rate of PEPCK carboxylation measured as 1A CO ? uptake. vn

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LIST OF FIGURES Figures Page 2-1 Examination of inorganic carbon substrate used by 46 Udotea for photosynthesis. DIC was added as mostly HCO," (I) with and without CA by adding NaHC0 3 solution to the reaction mixture. Of the 5 mM DIC present, 4.49 mM was HCO," and 0.03 mM was C0 2 DIC was added as CO, () with and without CA at a concentration of 5 mM by acidifying NaHC0 3 solution with HC1 in a syringe prior to adding it to the reaction mixture. 2-2 Measurement of photosynthesis in reaction mixtures 49 of pH 7.7
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3-5 Effect of temperature on purified PEPCK 88 carboxylation activity (•) at pH 6.8 and decarboxylation activity (o) at pH 7.8. 3-6 Influence of DIC concentration on PEPCK 89 carboxylation activity at the carboxylation optimum pH of 6.8 (•) and the decarboxylation optimum pH of 7.8 (o) 3-7 Competitive inhibition of PEPCK carboxylation 94 activity by 100 piM MPA with respect to PEP. (t) no MPA present, (o) with MPA present. 3-8 Competitive inhibition of PEPCK decarboxylation 95 activity by 100 pM MPA with respect to OAA. (•) no MPA present, (o) with MPA present. 3-9 Effect of of 4 mM malate on the K (DIC) for PEPCK 98 carboxylation. (•) no malate present, (O) with malate present. 3-10 The effect of 4 mM DHAP on K (DIC) for PEPCK 99 carboxylation. (•) no DHAP present, (o) with DHAP present. 3-11 Effect of 4 mM 3-PGA on PEPCK carboxylation with 100 respect to DIC concentration. (•) no 3-PGA present, (o) with 3-PGA present. 3-12 Effect of 4 mM F6P on the K m (OAA) for PEPCK 101 decarboxylation. (t) no F6P present, (o) with F6P present. 3-13 Effect of 4 mM F-1,6-BP on the K m (PEP) for PEPCK 102 carboxylation. (•) no F-1,6-BP present, (o) with F-1,6-BP present. 3-14 Effect of 4 mM F-1,6-BP on K_(DIC) for PEPCK 103 carboxylation. (•) no F-1,6-BP present, (o) with F-1,6-BP present. 3-15 Effect of 4 mM PEP on the K (OAA) for PEPCK 104 decarboxylation. (•) no PEP present, (o) with PEP present. 3-16 Effect of ADP on the K m (ATP) for PEPCK 105 decarboxylation. (•) no ADP present, (o) with ADP present. 3-17 Effect of 4 mM ATP on the K m (ADP) for PEPCK 107 carboxylation. (•) no ATP present, (o) with ATP present. 3-18 Proposed scheme for photosynthetic carbon 120 metabolism in Udotea. Carboxylation of PEP occurs by PEPCK in the cytosol. The OAA produced is rapidly reduced to malate by cytosolic MDH, thereby pulling the PEPCK reaction in the carboxylation direction. Malate moves to the chloroplast where it is decarboxylated, and the released COp is refixed by Rubisco. The acceptor molecule PEP is regenerated in the chloroplast by pyruvate P i dikinase. The PEP can then move back to the cytosol to accept another COp. IX

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ft ATPase AZ Bicine-NaOH BSA CA CAM CAMP CHES-NaOH Chi DBAZ DCMU DHAP DIC DIDS DTT DW ft EZ F-1,6-BP F6P Hepes-NaOH ^.5 LN 2 MDH KEY TO ABBREVIATIONS absorbance adenosine triphosphate synthetase acetazolamide n, n-bis ( 2-hydroxyethyl ) glycine-sodium hydroxide bovine serum albumin carbonic anhydrase Crassulacean acid metabolism cyclic adenosine monophosphate 2-(n-cyclohexylamino)ethanesulfonic acid-sodium hydroxide chlorophyll inorganic carbon dextran-bound acetazolamide 3( 3 4-dichlorophenyl ) -1 1-dimethylurea dihydroxyacetone phosphate dissolved inorganic carbon 4,4' -diisothiocyanatostilbene-2 2 -disulf onic acid dithiothreitol dry weight after acid treatment ethoxyzolamide fructose-1, 6-bisphosphate fructose-6-phosphate n( 2 -hydroxyethy 1 ) piperaz ine-n ( 2 ethanesulfonic acid-sodium hydroxide apparent Michaelis-Menten constant liguid nitrogen malate dehydrogenase

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MES MES-NaOH MPA OAA PCO PCR PEP PEPC PEPCK 3 -PGA PIPES-NaOH PMSF pro PVP-40 Rubisco RuBP Tris-HCl V max luCL 2-(n-morpholino)ethanesulfonic acid 2-(n-morpholino)ethanesulfonic acid-sodium hydroxide 3-mercaptopicolinic acid oxaloacetate photorespiratory carbon oxidation photosynthetic carbon reduction phosphoenolpyruvate phosphoenolpyruvate carboxylase phosphoenolpyruvate carboxykinase 3-phosphoglycerate piperazine-n,n' -bis (2-thanesulfonic acid)sodium hydroxide phenylmethylsulfonyl fluoride protein polyvinylpyrrolidone with molecular weight of 40,000 kilodaltons ribulose-1 5-bisphosphate carboxylase/oxygenase riubulose-1, 5-bisphosphate tris ( hydroxymethyl ) aminomethane-hydrochloric acid maximum velocity micro Curie XI

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Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy INORGANIC CARBON UTILIZATION AND THE ROLE OF PHOSPHOENOLPYRUVATE CARBOXYKINASE IN THE PHOTOSYNTHESIS OF THE MARINE MACROALGA UDOTEA By Beth Ann Burch December 1993 Chairman: George Bowes Major Department: Botany Udotea is a marine, green, siphonaceous macroalga with C,-like photosynthetic characteristics. At pH 8.2, 15 mM dissolved inorganic carbon (DIC) was required to saturate photosynthesis, which is 7-fold greater than the concentration in natural seawater. Carbon dioxide was the preferred DIC species for photosynthesis. Acidification of the thallus surface occurred in the light and the dark, which could produce C0 2 for diffusion into the siphons. External carbonic anhydrase activity was lacking. In pH drift experiments, the highest final pH value attained was only 8.95, which is in the range typical of C0 2 ~only users. However, some HCO,~ use was indicated by enhanced photosynthesis when the HC0 3 ~ concentration was increased at a given C0 2 concentration. Photosynthesis was inhibited 13 to 33% by 4, 4 -diisothiocyanostilbene2,2 'disulphonate and 34% by the plasma-membrane ATPase inhibitor vanadate, which also halted thallus acidification. Thus, ATP may have a role in the DIC uptake process. Calcium chloride was necessary for photosynthetic DIC uptake, but not 2 evolution. This is consistent with calcification by Udotea., as 50% of the dry weight of the alga was composed of carbonate salts.

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The kinetics of isolated phosphoenolpyruvate carboxykinase (PEPCK) were analyzed to determine how this enzyme operates for carboxylation in Udotea photosynthesis. Unlike carboxylation by ribulose-1, 5bisphosphate carboxylase/oxygenase, the PEPCK carboxylation reaction was 2 insensitive though C0 2 was the substrate. The carboxylation pH optimum of 6.8 was lower than for decarboxylation, and at this pH, which is close to the probable pH of the cytosol, where PEPCK appears to be located, the carboxylation V was nearly 2-fold greater than that of decarboxylation. For carboxylation, the K m values for C0 2 phosphoenolpyruvate (PEP), and ADP were 2.1 mM, 1.7 mM, and 30 pM, respectively; for decarboxylation, the K values for oxaloacetate (OAA) and ATP were 35 and 2 ^M. Dihydroxyacetone phosphate, 3phosphoglycerate, and ATP inhibited carboxylation, while fructose-1, 6bisphosphate stimulated it by lowering the K (PEP) from 1.6 mM to 0.5 mM. Fructose-6-phosphate and C0 2 inhibited decarboxylation. These observations raise the possibility of metabolite regulation of the carboxylation/decarboxylation ratio. Both reactions were competitively inhibited with respect to PEP and OAA by 3-mercaptopicolinic acid. The carboxylation rate was enhanced 6-fold in the presence of malate dehydrogenase isolated from Udotea. X1JLX

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CHAPTER 1 GENERAL INTRODUCTION AND LITERATURE REVIEW The Marine Macroalga Udotea Habitat and Morphology Udotea conglutlngata (Ellis and Solander) Lamouroux and U. flabellum (Ellis and Solander) Lamouroux used in this investigation are marine, green, calcified, macroalgae that belong to the Family Udoteaceae of the Order Caulerpales, Class Chlorophyceae and Division Chlorophyta (20, 153). These algae can be found in tropical to subtropical marine waters (21), growing in soft mud or calcareous sand, often among seagrasses (45). The thallus of Udotea is composed of intertwining siphons with cell walls of 0-1,3-xylan (21, 23), which exists in a hollow helix conformation (97). No cellulose was detected. Thalli are heteroplastidic with photosynthetic chloroplasts and starch-storing amyloplasts (153). Chloroplasts of Udotea have lamellae with various numbers of thylakoids and a thylakoid-organizing body of concentric double membranes at one end (153). The accessory photosynthetic pigments siphonein and siphoxanthin are also found in members of this family (153). In adult thalli, amyloplasts are found further down in the thalli than the chloroplasts (24). Sexual reproduction in members of the Udoteaceae seldom happens (21), but when it does occur, diploid dioecious thalli produce haploid gametes in a holocarpic manner, where an entire siphon is converted from the vegetative state to one where the nuclear division products are haploid gametes (101, 153). Gametes are released into the water when the siphon bursts open at the tip, and thallus death then follows (21). Anisogamous germ cells of two opposite mating types fuse, producing a

PAGE 15

2 diploid zygote, which then develops into a protosphere, which has starch-containing chloroplasts (21). Upon maturation, the new thallus that develops from the protosphere becomes heteroplastidic. Vegetative reproduction, which is the most frequent type of reproduction in Udotea, occurs when new thalli arise from a branched rhizoidal system (21). These habit and reproductive features of Udotea indicate that it possesses a number of derived and highly derived characteristics. Although its evolutionary development was in a different direction from that which eventally led to the vascular land plants, Udotea is by no means a "primitive" alga; rather, it belongs to a group considered "most derived (153)." Calcification Udotea conglutlnata and U. flabellum are calcified representatives of this genus. The calcification mechanism in algae is tied closely with photosynthesis, as no CaC0 3 is deposited until the plant is photosynthetically active (22, 23, 24). The mechanism of calcification in these two species differs slightly in that U. conglutlnata deposits aragonite crystals of CaCO, intracellular^ within organic layers of a sheath that begins at the cell wall and forms an outer wall of the siphons (19). Some deposits also occur in the intersiphonous areas of the thallus. In addition, crystals of calcium oxalate are deposited in the vacuole in a randomly oriented fashion (56). In contrast, U. flabellum lacks calcium oxalate crystal deposits in its vacuoles (56). At the outer surface, the siphons are highly branched, forming a developed cortical surface (19). In this cortex region, CaC0 3 is deposited between layers of the siphon wall, while away from the exterior surface, CaC0 3 aragonite crystals are deposited in the inter-siphon spaces (19). Both primary and secondary crystal deposits occur in the cortex region, and this pattern is somewhat similar to what occurs in sheath calicif ication, exhibited in U. conglutlnata (19).

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3 Thus, a. flabellum exhibits both intraand intercellular calcification (19). Regardless of where CaC0 3 precipitation occurs, this mechanism requires an enclosed space where a high pH can be generated, in this case, by the removal of C0 2 for use in photosynthetic carbon assimilation (23). It has been suggested that the Ca 2+ for precipitation in calcareous macroalgae comes from an intracellular pool (98). The trans-membrane Ca 2+ transport may be achieved by an ATPase that exchanges 2 H + per Ca 2+ thus helping to generate the external high pH needed for CaC0 3 precipitation (98). Photosynthesis Terrestrial plants have been divided into four categories based on the biochemistry of their photosynthetic mechanism. These categories are C 3 C^, C^-C^ intermediates, and Crassulacean acid metabolism (CAM). The categories do not follow taxonomic lines, as one genus contains representatives from three of the four photosynthetic categories. The C 3~ C 4 intermediate category will not be discussed. C ; Photosynthetic Category By far the vast majority of plants in the North American flora fits into the C 3 category. The leaf mesophyll of plants in this group is differentiated into a palisade layer on the adaxial side and a spongy layer on the abaxial side. Chloroplasts are dispersed throughout the mesophyll layer and are usually lacking in the bundle sheath. The chloroplasts are all the same, i.e., the thylakoids are stacked into grana, have both photosystems I and II, and have the full complement of photosynthetic carbon reduction (PCR) cycle enzymes. Of the seed plants, both gymnosperms and angiosperms (including monocots and dicots) are represented, so vascular bundle arrangement varies somewhat. Stomates are found on both leaf surfaces, although usually more prevalent on the abaxial surface. Guard cells are functional and open

PAGE 17

4 during the daytime to facilitate the exchange of C0 2 2 and water vapor between the leaf and the surrounding atmosphere (51). The C 3 plants use the PCR cycle to fix atmospheric carbon into carbohydrate. Ribulose 1, 5-bisphosphate carboxylase/oxygenase (Rubisco) is the predominant carboxylating enzyme, and it incorporates atmospheric C0 2 directly into 3-phosphoglycerate (3-PGA). Because of the active site conformation of Rubisco, which allows either C0 2 or 2 to react with ribulose 1, 5-bisphosphate (RuBP), the enzyme also catalyzes the first step in photorespiration or the photorespiratory carbon oxidation (PCO) cycle. Rubisco must be activated by C0 2 and Mg 2+ to be catalytically competent (32, 89, 92). Carbon dioxide, rather than HC0 3 or C0 3 is the carbon species utilized for both activation and catalysis, and the activator and catalytic C0 2 molecules are distinct according to kinetic and spectroscopic measurements (75, 89). A C0 2 molecule and Mg 2+ cation must bind to each of the eight catalytic sites for Rubisco to be completely activated (81). Light affects Rubisco in two important ways: light induces changes in the chloroplast such as increasing the stromal pH from around 7 to approximately 8.2 (92) and increasing Mg 2+ concentration in the stroma by about 3 mM (92). These changes increase enzyme activity (32) and activate Rubisco via Rubisco activase. The binding of C0 2 and Mg 2+ to the catalytic sites of the enzyme is facilitated (129, 154). In the PCO cycle, RuBP is oxidized to 3-PGA and 2-P glycolate, the latter of which is dephosphorylated in the chloroplast, and the resulting glycolate enters the peroxisome, where it is metabolized to glycine. Glycine then migrates to the mitochondrion where C0 2 is released in the conversion to serine (91). The PCO cycle decreases net carbon fixation by 20 to 40% (16) because 2 directly competes for C0 2 at the Rubisco active site. This photorespiration decreases the amount of RuBP available for carboxylation, and one third of the fixed C0 2 is released. The C0 2 compensation point, the concentration of CO ? at which

PAGE 18

5 the net carbon exchange in the light is zero, is achieved when the C0 2 released by the PCO cycle just balances that reduced by the PCR cycle. For terrestrial plants, the C0 2 released in photorespiration is a large component of the CC> 2 compensation point, but the CC> 2 released by dark respiration is not. When the plants are placed into 1% o 2 where photorespiration is all but eliminated, measurable respiratory C0 2 release is virtually zero (54). The operation of the PCO cycle causes the C0 2 compensation points in C 3 plants to be quite high, in the range of 30 to 70 ^L C0 2 IT 1 (16). Photorespiratory C0 2 release is energetically expensive because C0 2 must first be reduced to RuBP with NADPH and ATP produced in the light reactions before oxidation to glycolate can occur (90), and the process itself requires NADH and ATP to function. The release of NH 3 and its subsequent refixation is also a costly process. Evidence that both carboxylation and oxygenation occur at the same site includes 1) inhibition of the two processes by carboxyarabinitol 1,5-bisphosphate (similar in structure to the C 6 intermediate formed immediately after carboxylation of RuBP), 2) activation of both functions to the same degree by C0 2 and Mg 2+ (91), and 3) demonstration that K m (C0 2 ) equals K^Og) and K m (0 2 ) equals K^COg) (26). However, although the ratio of C0 2 to oxygen is relatively constant at all temperatures, oxygenation is favored over carboxylation at the higher temperatures because C0 2 is less soluble in water than 2 under these conditions (65, 86, 107), and Rubisco has a lower affinity for C0 2 at higher temperatures (104, 132). Therefore, the optimum temperature for C 3 photosynthesis ranges from 15 to 25C (16). C, Photosvnthetic Category In some terrestrial plants, an additional metabolic pathway has arisen which serves to concentrate C0 2 in the vicinity of the Rubisco active site, thereby decreasing photorespiration (48). The origin of the C 4 photosynthetic mechanism appears to be polyphyletic, and for

PAGE 19

6 terrestrial plants it is found only in the more advanced angiosperms (115). It occurs most frequently in herbaceous plants including annual and perennial herbaceous monocots and dicots. Plants in this photosynthetic category exhibit Kranz anatomy in which the bundle sheath surrounding the vascular tissue is distinctive and composed of large, closely packed cells that are larger than the mesophyll cells. Chloroplasts in the bundle sheath are arranged specifically in accord with the type of C A metabolism the plant uses (48). Plants of the NADP-malic enzyme type contain dimorphic chloroplasts (48). Those of the mesophyll cells have grana, pyruvate P. dikinase, and relatively high levels of NADP-malic dehydrogenase, but lack Rubisco and have the enzymes of only the reductive phase of the PCR cycle. However, the Kranz cell chloroplasts lack grana but have Rubisco and all of the PCR cycle enzymes (48). All C 4 plants have two carboxylation reactions. In the first reaction, HCOj" is the form of dissolved inorganic carbon (DIC) used. Carbonic anhydrase (CA) facilitates the equilibrium between this species and atmospheric C0 2 Phosphoenolpyruvate is carboxylated with HC0 3 by phosphoenolpyruvate carboxylase (PEPC), resulting in the production of the C 4 acid oxaloacetate (OAA) Oxaloacetate is either reduced to malate or transaminated to aspartate, which is then transported to the bundlesheath cells. Either malate is decarboxylated directly or aspartate is deaminated and reduced to malate, which is then decarboxylated by one of three enzymes (48). In the NADP-malic enzyme subgroup of C^ plants, which includes Zea mays and Saccharum offlcinarum, OAA from carboxylation in the mesophyll cell cytosol is reduced to malate and then translocated to the bundle sheath cell chloroplasts, where the malate is oxidized and decarboxylated to pyruvate and C0 2 The pyruvate is transported back to the mesophyll cell chloroplasts, where it is phosphorylated to PEP by pyruvate P i dikinase. The CQ 2 is then refixed by Rubisco in the

PAGE 20

7 chloroplasts of the bundle sheath cells. Chloroplasts in the bundle sheath cells of these species are agranal and deficient in photosystem II activity (48). They are located near the centrifugal wall of the bundle sheath cells. The predominant transport metabolites in this subgroup are malate and pyruvate (48). In the NAD-malic enzyme subgroup, exemplified by Panicum mlliaceum and Amaranthus retroflexus, OAA from PEPC carboxylation is transaminated to aspartate in the cytoplasm of the mesophyll cells and then transported to the mitochondria of bundle sheath cells. Aspartate is deaminated to OAA in the mitochondria and then reduced to malate, which is oxidized and decarboxylated to pyruvate and C0 2 Pyruvate is transaminated to alanine in the bundle sheath cell cytosol. Alanine moves back into the mesophyll cell cytosol and is deaminated to pyruvate, which them moves into the chloroplasts, where phosphorylation occurs via pyruvate P i dikinase to regenerate PEP. The liberated C0 2 is refixed by Rubisco in the chloroplasts of bundle sheath cells. Chloroplasts of the bundle sheath cells in these species do have grana and are located in the centripetal position in the cells. These plants also have many more large mitochondria in the centripetal position of the bundle sheath cells compared to other groups. Here the major transport metabolites are aspartate and alanine (48). Plants in the PEPCK subgroup, such as Panicum maximum and Chloris gay ana, transaminate the OAA produced by PEPC to aspartate in the cytoplasm of mesophyll cells, and aspartate is then moved to the cytosol of bundle sheath cells. Here deamination produces OAA which is decarboxylated to pyruvate followed by ATP-dependent formation of PEP in the cytoplasm. The PEP is dephosphorylated to pyruvate, which is then transaminated to alanine. Alanine then moves into the cytosol of the mesophyll cells. Alanine is deaminated to pyruvate which enters the chloroplasts and is phosphorylated to PEP via pyruvate P. dikinase. As above, the C0 2 is refixed by Rubisco in the bundle sheath cell

PAGE 21

8 chloroplasts. These plants have granal chloroplasts toward the centrifugal position in the bundle sheath cells. The major transport metabolites in this subgroup are also aspartate and alanine (48). In the C 4 photosynthetic mechanism, less than 5% of the C0 2 assimilated from the atmosphere enters 3-PGA directly. Instead, the C0 2 that enters the PCR cycle comes almost exclusively from the decarboxylation of the organic acids; essentially Rubisco is isolated from the atmosphere (48). This arrangement is beneficial for the plant in that the C0 2 concentration is elevated or concentrated. Photosynthesis by C 4 plants is independent of 2 concentrations ranging from 2 to 21% (115). Because the C0 2 /0 2 ratio is increased in C 4 plants, the limitations imposed by C0 2 and 2 solubility characteristics with increasing temperature are overcome (86), which has a role in elevating the temperature optimum in these species into the 30 to 47C range (16). The function of the elaborate C^ acid system is to concentrate C0 2 and in doing so, essentially eliminates the photorespiratory release of C0 2 However, some 2-P glycolate does form, and there is carbon flux through the glycolate pathway (108). A measurable indicator of less C0 2 release from the leaf is the very low C0 2 compensation point measured for C^ plants, usually in the range from to 10 pL C0 2 L (16). Dark respiration does not constitute any measurable component of the C0 2 compensation point, as evidenced by these low values. The C^ acid mechanism, then, is an effective means by which to reduce photorespiratory C0 2 release. Although less C0 2 is photorespired in the C 4 pathway, thereby reducing energy loss in the form of NADPH and ATP used to produce RuBP and energize the PCO cycle, C 4 plants do reguire an additional two or three ATP molecules to fix C0 2 because of the last step of the seguence where pyruvate is converted to PEP by pyruvate P i dikinase (48). Under conditions of higher temperature and irradiance, C^ plants have a major advantage over C 3 plants in that C A plants are not affected by the

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9 decrease in C0 2 solubility with increased temperature because of the C0 2 concentrating mechanism (48). However, under lower irradiance levels and temperature, C 3 plants can match or even exceed the net carbon gain exhibited by C 4 plants (48). CAM Photosvnthetic Category Crassulacean acid metabolism can actually be considered a form of C 4 photosynthesis. Atmospheric C0 2 is converted to HC0 3 ~ by CA and fixed by PEPC into OAA, which is then reduced to malate. Decarboxylation then occurs by either NAD-malic enzyme or PEPCK, and the liberated C0 2 is fixed into carbohydrate by Rubisco. Obligate CAM species utilize the CAM pathway for carbon fixation all the time, while facultative CAM species can shift dramatically the physiology and biochemistry of carbon fixation from C 3 to CAM mode. Water availability determines the photosynthetic metabolism; low availability induces CAM (144). These plants exhibit variable C0 2 compensation points. During the early part of the day, during Phase III of the CAM cycle, in which the malate stored at night is decarboxylated, the C0 2 compensation point is low because the decarboxylation concentrates C0 2 in the vicinity of Rubisco. The C0 2 level may rise to be greater than 1% or 10,000 pL L" 1 Although the 2 concentration increases as photosynthesis proceeds, the favorable C0 2 /0 2 ratio limits photorespiration (144). In addition, the difference in K m (C0 2 ) and K m (0 2 ) for Rubisco favors C0 2 fixation. However, toward the end of the day, during Phase IV when all of the stored malate has been depleted, the stomates open, and the plant carries on C 3 photosynthesis. The C0 2 compensation point in this phase has been measured at 50 f/L C0 2 IT 1 and a burst of C0 2 results when plants in this phase are artificially placed in the dark; both characteristics point to an active photorespiration process. Although CAM species can photorespire, the C0 2 released can serve as an internal carbon source for photosynthesis (111). But unlike C 4 plants, CAM species lack Kranz anatomy (59). PEPC and Rubisco are located in the same cells, but their

PAGE 23

10 activities are temporally separate; fixation by PEPC at night results in the storage of malate in the vacuole until daylight when malate transport and decarboxylation and subsequent fixation of the released C0 2 by Rubisco begins (113). Plants with this photosynthetic pathway usually occur in arid habitats, where the mechanism aids in water conservation. However, Isoetes howellli, a submersed aquatic plant, also exhibits this type of carbon metabolism (83) as a means of overcoming the carbon limitations in its environment. Marine Algal Photosynthesis In numerous marine macroalgae studied thus far, photosynthesis is characterized by 2 ~insensitive photosynthesis, low C0 2 compensation points, and low photorespiration rates, but some species do not share these attributes (77). Such characteristics make the algae photosynthetically more similar to the terrestrial C^ plants than to the C 3 species. This feature is intriguing in that the algae are considered to be "primitive" among plants. The C, photosynthetic mechanism is considered "advanced," as it involves specialized anatomy and compartmentalized enzymes with ample shuffling of metabolites between cells. One wonders how these "primitive" plants can utilize such an "advanced" form of metabolism. In contrast, Raven and Osmund (119) have concluded that some subtidal macroalgae, including the green Codlum fragile, and the browns Dlctyota dichotoma, Padina vlckersii, Sargassum fillpendula, and Fucus vesiculosus, possess photosynthetic capacity that cannot be used during steady-state, light-saturated photosynthesis unless the inorganic carbon concentration is increased, a scenario reminiscent of the terrestrial C, species. Thus, the inorganic carbon concentration in marine waters is not sufficient to saturate photosynthesis for some algae, just as the atmospheric concentration of C0 2 does not saturate photosynthesis in the terrestrial C, species. One of the challenges a plant faces in a marine environment is obtaining inorganic carbon for photosynthesis. Because dissolved gases

PAGE 24

11 diffuse about 10,000 times more slowly in water than they do in air (25), a submersed plant must be able to overcome this physical limitation in its environment. A survey of marine macroalgae across the algal divisions indicates that the DIC concentration in seawater is too low to overcome the effects of 2 on photosynthesis unless the alga employs some sort of carbon concentrating mechanism (71). Such mechanisms may involve direct HC0 3 ~ uptake or C 4 acid metabolism. With regard to the particular species of DIC utilized by an alga, one must consider the influence of the pH and salinity of seawater on the proportion of each of the inorganic carbon species. The pH of seawater is in the 8.0-8.2 range and the salinity around 35 parts per thousand. Under these conditions, with the water in equilibrium with the atmosphere, the predominant species is HC0 3 ~, present at a concentration of about 2 mM. Only 10 /jM of the total carbon is C0 2 (133). It has been determined that C0 3 2 is not a photosynthetically active species, even when its relative proportion dramatically increases, as may occur in a rockpool containing photosynthesizing algae (93), so further discussion will focus on only HC0 3 ~ and C0 2 The macroalgae examined show a range of affininities for HC0 3 ~ (94). A common method of assessing this ability is based on measuring the photosynthetic rate and comparing it to the rate expected based on the C0 2 produced as a result of HC0 3 dehydration (77). If the observed photosynthetic rate exceeds the spontaneous C0 2 supply rate, the species likely is using HC0 3 in addition to C0 2 (77). However, before a specific HC0 3 ~ utilization mechanism can be hypothesized, a lack of external CA must be demonstrated. This enzyme located outside the plasma membrane facilitates the conversion of HC0 3 ~ to C0 2 or vice versa, depending on the pH. The C0 2 can diffuse across the membrane into the cell (77), while HC0 3 ~ cannot because of the charge on the molecule. Based on this criterion, most marine macroalgae studied have been found to need HC0 3 in addition to the C0 2 spontaneously supplied

PAGE 25

12 by dehydration to support the observed photosynthetic rate (6, 77), and the algae show a consistently high capacity for inorganic carbon transport (14). The inorganic carbon diseguilibrium technique can also be used to ascertain the species of inorganic carbon preferred by a plant. The plant is given either CQ 2 or HC0 3 ~ under conditions where the time for equilibrium to be established between the two carbon species is prolonged by low temperature. Based on whether photosynthesis occurs more rapidly in the presence of either C0 2 or HC0 3 ~, this rate difference indicates which particular carbon species is preferred by that plant (77) Of course, the enzymes involved in inorganic carbon utilization are a key component of the photosynthetic mechanism. In all macroalgae examined, Rubisco has been determined to be the primary carboxylase (11, 71) In addition, the variability in photosynthetic rates observed for the marine macroalgae correlates well with the measured carboxylase activity (14), even to the extent that the photosynthesis to carboxylase activity ratio is close to one. Rubisco activity plays a role in regulating the photosynthetic capacity where internal CO, concentration is high and photorespiration is suppressed, but other factors also have a role in limiting the photosynthetic rate under such conditions (14). Chlorophvta Nearly all green macroalgae examined to determine their photosynthetic mechanism exhibit a C,-type pattern, where the initial and primary carboxylating enzyme is Rubisco (11, 71, 77, 123). Studies with CO-, have shown that the radioactivity first appears in 3-PGA, the first product of Rubisco carboxylation. Only later, during the chase time, does the C appear in the C, organic acids malate and aspartate, thereby indicating that these compounds are produced later in the metabolic sequence rather than being early products that are rapidly turned over, losing the "C to other molecules (123).

PAGE 26

13 The green macrophyte Prasiola stlpitata, which grows in the veryhigh splash zone, has a low C0 2 compensation point and the photosynthetic rate is saturated at the seawater concentration of inorganic carbon (118). External HCO,~ is used for photosynthesis and no CAM-like features occur (118). One green macrophyte studied extensively is Ulva. Some species of this ubiguitous alga move HCO, across the plasma membrane into the cytoplasm, which saturates photosynthesis at the inorganic carbon (C. ) concentration found in natural seawater (10). Despite a low C0 2 compensation point and Op-insensitive photosynthesis, Ulva is biochemically a C 3 plant. Activity of PEPC is detectable, but the level is just one-tenth that of Rubisco. The data from radioactive labeling studies corroborate this finding; the first compound labeled with C is 3-PGA, and the radioactivity subsequently appears in sugars (11). Thus, it is suggested that a carbon concentrating mechanism based on HCO^ uptake is responsible for these features (11). During photosynthesis, the entire surface of U. fasciata reaches pH 10; no zones of acidification such as occur in the green alga Chara were detected (12). For U. lactuca, no carbonic anhydrase activity could be measured external to the cytoplasm (12, 47). When the CA is inhibited by acetazolamide (AZ), C0 2 from the unstirred layer cannot support the observed photosynthetic rate; thus, it is concluded that HC0 3 ~ uptake occurs, although C0 2 use at times cannot be excluded (12). Therefore, HCO,~ is the preferred species transported in U. lactuca (13). The mechanism for this movement could be HC0 3 /H antiport or HC0 3 ~/OH~ symport (12, 47). It is hypothesized that HC0 3 ~ is transported across the plasma membrane and then converted intracellularly to CO ? which moves to the chloroplast and can then be used by Rubisco for catalysis. Whether the HC0 3 ~ translocation occurs via facilitated diffusion as occurs in red blood cell membranes or by active transport remains to be determined (47). In U. fasciata, the

PAGE 27

14 internal carbon concentration can reach 2.3-6.0 mM with the bulk concentration of the surrounding medium at 0.02-1.5 mM, which indicates that active C i accumulation occurs (13). With a cytoplasmic pH of 7.2 and high internal CA activity, the internal C0 2 concentration could be high enough to suppress photorespiration and saturate carboxylation, even though this Rubiso has K m (C0 2 ) of 70 pM (13). A slightly different mechanism for C i uptake may occur in U. rlglda. This alga has both an external and internal CA (15). HC0 3 ~ is converted to C0 2 outside the cells, and C0 2 passes through the plasma membrane. Support for this mechanism comes from studies with dextranbound AZ (DBAZ), which inhibits only external CA. In the presence of DBAZ, photosynthesis is inhibited at pH 8.6 when HC0 3 is virtually the only inorganic carbon source, but no inhibition occurs at pH 6.5, where ample CD 2 is present (15). However, active HC0 3 ~ transport cannot be excluded based on these data (15). In addition, the external alkalinization of the thallus is inhibited in the presence of DBAZ. Ethoxyzolamide (EZ) also inhibits photosynthesis, at both pH 6.5 and pH 8.6; however, adding C0 2 to the medium at pH 8.6 overcomes the inhibition (15). The internal CA plays an important role in the C i utilization mechanism in this alga. Photosynthesis in Codlum decortication appears Cj-like with 2 ~ sensitive photosynthesis and minimal PEPC and PEPCK activities (123). The high C0 2 compensation point is also sensitive to 2 The DIC in natural seawater does not saturate photosynthesis, but saturation occurs at 5 mM DIC (123, 124). Both the cytoplasm and isolated chloroplasts have CA activity, and some of the chloroplast activity is associated with the membranes (123). There is also a report of extra-cellular CA activity in Codlum (40). In addition, higher photosynthetic rates can be obtained with the addition of HC0 3 ~ to the photosynthetic medium, which indicates that the alga is able to use HC0 3 (124), as is also reported for C. fragile (119).

PAGE 28

15 The green microalga Stlchococcus bacillaris has high CA activity and a high affininty for COp. In a medium of pH 5.0, the cells can concentrate internal C0 2 to a level 20 times greater than that in the bathing medium, and this concentrating does not occur at pH 8.3 (102). Therefore, Stlchococcus is a COj-user, and it is suggested that this concentration is accomplished via an active COp transport mechanism (102). A green macroalga with photosynthetic metabolism that stands in stark contrast to other green algae investigated is Udotea flabellum. However, U. flabellum exhibits Op-insensitive photosynthesis and a low C0 2 compensation point, both of which suggest a low level of photorespiration (123), similar to a number of the green algae. A comparison of photosynthetic rates measured at pH 5.0 and 8.0 indicates that the rate is higher at the higher pH, which suggests that the alga is able to use HC0 3 for photosynthesis (123). At pH 5.0, EZ has a limited effect on photosynthesis, but at pH 8.0, the rate decreases 39%, which shows that CA activity has a role in Udotea photosynthesis (123). Examining photosynthesis in this alga with H^CO," and with and without 3-mercaptopicolinic acid (MPA) an inhibitor of PEPCK, indicates that decreased label is found in the photorespiratory products when PEPCK is operating and that the C, organic acids malate and aspartate are early products that rapidly lose their radioactivity (123). Such a mechanism is reminiscent of the metabolism of the terrestrial C, species. However, in those plants, the initial carboxylation reaction is catalyzed by PEPC, and the activity of this enzyme in U. flabellum was barely detectable (123). PEPCK has substantial activity, though, such that it rivals Rubisco activity (123). Inhibiting the PEPCK activity with MPA causes an increase in the photorespiratory intermediates along with a decrease in the radioactivity found in malate and aspartate (123).

PAGE 29

16 Carbonic anhydrase activity in this alga may be related to HC0 3 ~ use, but it is not a component of the mechanism to overcome the ? inhibition of photosynthesis. Rather, it appears that PEPCK activity may be the key to reduce photorespiration. In contrast to other algae, it is doubtful that the |3-carboxylation catalyzed by PEPCK functions for anapleurosis. Instead, the C^-like metabolism depends on active PEPCK because inhibiting the activity with MPA decreases the photosynthetic rate by 70%, increases the Warburg effect, reduces the C, acid label by 66%, and increases label in the PCO cycle intermediates by 3-fold (122, 123). These effects of MPA on photosynthesis cannot be explained by an HC0 3 utilization mechanism in U. flabellum, nor is it likely that the effects of MPA place PEPCK in the role of simply refixing photorespired C0 2 ; the effects are too large for this to be the case (122). Also, in the absence of DIC, exogenously-supplied malate is able to stimulate photosynthesis, but if MPA is present, no such stimulation occurs (122). Because Kranz anatomy associated with terrestrial C, species is not present in Udotea, a C^-type mechanism would have to involve an organellar separation of events (122). This type of separation has been reported for the freshwater angiosperm Hydrilla verticillata, which also lacks Kranz anatomy (25, 27). A parallel function between PEPCK of Udotea and PEPC of terrestrial C, species is suggested; the activities of other enzymes typically associated with C, metabolism are present in Udotea at high enough levels that it is reasonable to assume that C, photosynthetic biochemistry could occur (122, 123). Thus, for Udotea, the evidence appears quite convincing that a type of C^ biochemistry occurs which is dependent on PEPCK activity. However, this mechanism has not been reported for any other marine macroalgae. Phaeophvta The photosynthetic characteristics of the brown algae investigated display a mixture of features. For example, Turbinarla turblnata and Lobophora varlegata exhibit a Warburg effect on photosynthesis at 0.5

PAGE 30

17 and 2.5 mM DIC, respectively (71). Although Turbinarla has a detectable malate pool, it does not appear to function in a CAM-like mechanism (71). Another brown, Dllophus gulneensls, has high PEPC and PEPCK acitivities, but Rubisco is the major carboxylating enzyme (71). Of the brown algae surveyed by Kremer and Kuppers (85), the PCR cycle is the only light-dependent carboxylation system, and greater than 90% of the early photosynthetic products are phosphorylated compounds, with 3-PGA being predominant, thereby lending support to the assertion that these plants operate C 3 photosynthesis (85). They suggest that when PEPCK activity is high, the enzyme functions for light-independent carbon fixation. In addition, no PEPC activity is reported for the species they examined (85). However, in contrast to Turbinarla and Lobophora, the eulittoral brown algae appear C 4 ~like in their photosynthetic features: low C0 2 compensation points, and 2 -insensitive photosynthesis (79, 141), which suggests that an inorganic carbon concentrating mechanism is present in these algae. In addition, photosynthesis is saturated with inorganic carbon at the concentration of 2 mM found in natural seawater (141), so these algae are able to photosynthesize faster than would be expected based on the dehydration rate of HCOj" alone (79). Some Fucaceae, of which Ascophyllum nodosum is a member, for example, have C 4 ~like physiology but C 3 -like biochemistry. They demonstrate an active membrane transport of C0 2 and/or HC0 3 ~ although the location of the transporter, whether at the plasma membrane or chloroplast membrane, remains to be determined (78, 141). A. nodosum has a H buffering system that is independent of the ion exchange in the cell wall (5). This alga also has a photosynthetic buffering capacity connected to the H + buffer, which is an adaptation to the alga's growing in the intertidal region. These buffering systems enable the plant to produce C 2 in excess of the inorganic carbon taken up for photoreduction (5). A. nodosum has high PEPCK activity, which may be involved in

PAGE 31

18 producing the pool of C0 2 -exchanging organic acids that function as the photoreductant buffer (5) as a complement to or an integrated part of a CAM-like system (6). The benefit of these two buffering systems to the alga is that they allow some photosynthetic electron transport to be maintained, even when exogenous carbon is in short supply. These systems could afford protection against photoinhibition (5). When CA is inhibited in A. nodosum, the C0 2 compensation point increases 25-fold and the apparent K Q5 (C0 2 ) for photosynthesis increases 8-fold, resulting in a shift of photosynthetic gas exchange characteristics from C 4 -like to C 3 -like (77). The rate of photosynthesis eguals the rate of C0 2 production and diffusion from the bulk phase when external CA is inhibited. Therefore, it appears likely that C0 2 is the species assimilated, and the C^ physiological characteristics depend in part on the operation of an external CA (77). Two other species hypothesized to have an active HCOj" uptake mechanism are Fucus distichus and Pelvetlopsis limit at a, which have no external CA, but have photosynthetic rates that exceed the photosynthetic rate possible based on spontaneous HC0 3 ~ dehydration alone (42). Some algae of the Laminariales, including Laminaria digitata, L. hyperborea, L. saccharina and Alaria esculenta, and some others of the Fucales, such as Halidrys siliguosa, have some C,-like gas exchange characteristics, but the C0 2 compensation points are higher than found in the Fucaceae, yet all have measurable external CA activity (141). These species appear to be less avid "HC0 3 users," but what features they do exhibit are probably due to active inorganic carbon influx as part of a concentrating mechanism (141). Both Fucus and Laminaria exhibit light-dependent external alkalinization of the surrounding medium, and this process is inhibited by adding DCMU, a light reaction inhibitor that blocks electron flow between guinone and the B protein, to the systems at pH 8.0 but not at pH 6.0 (64). Species of both genera have an external CA, as AZ inhibits

PAGE 32

19 the alkalinization at pH 8.0 (64). The proposed mechanism for Cuptake is that an external CA converts HC0 3 from the surrounding medium to CO ? which then moves into the cells for photosynthesis. It is not known whether C0 2 movement occurs by diffusion only or whether an active uptake process is involved (64). The site of C0 2 transport has yet to be determined; it may be at the plasma membrane and/or the chloroplast envelope. Carbon dioxide is pumped from the external medium into the cytosol by using energy from photosynthetic electron flow rather than from mitochondrial respiration (140). Another study of Laminaria dlgltata and several other species of brown algae that are C i -limited in natural seawater showed that when photosynthesis is saturated by red light, a blue light pulse stimulates the photosynthetic rate (55). The effect is hypothesized to involve an increase in the C0 2 supply to the plant; whether this is an effect on the external CA or H + efflux pump in the plasma membrane or on the carbon-fixing enzymes in the chloroplast remains to be determined (55). Photosynthesis in Dlctyota dichotoma, Padlna vlckerslae, Sargassum flllpendula, and Fucus veslcuolus can use HCC> 3 ~, at least at pH 9.4 and 2 mM DIC (119). Of the four taxa, F. veslcuolus is able to take up HC0 3 effectively and is carbon saturated at ambient concentration of DIC, similar to A. nodosum (77). Members of the Phaeophyceae are noted for exhibiting high PEPCK activity, but these organisms biochemically exhibit C 3 -like photosynthesis with 3-PGA appearing as the first product (85). Subseguent studies using 14 C show a large amount of the radioactive carbon is located in malate, and then the malate is rapidly metabolized to mannitol (85). In F. veslcuolus, a substantial portxon of C appears in malate and aspartate, thus suggesting active P-carboxylation. However, rapid turnover of these organic acids does not occur, indicating that these compounds are really metabolic endproducts (85). In addition, Rubisco from A. nodosum has a carboxylase/oxygenase activity ratio of about six, which is similar to

PAGE 33

20 that found in higher plants with C 3 photosynthesis (77). For a long time, the PEPCK enzyme has been said to function in light-independent carbon fixation. However, in A. nodosum, dark fixation of carbon does not exceed 5% of the light fixation rate, and usually the dark fixation rate is much lower (77). Thus, the role of PEPCK in this group remains unclear, but it is suggested that DIC accumulation accounts for the observed gas exchange features that make some algae in this group appear C 4 -like in their metabolism (77). It is currently believed that a biochemically based C 4 -like photosynthetic metabolism is not widespread among the algae (77). Rhodophyta An investigation of photosynthesis in the red algae indicates that more than 90% of the early photosynthetic products are phosphorylated compounds, with 3-PGA constituting the largest proportion (85). These data support Rubisco as the major carboxylating enzyme, suggesting that the red alga incorporate inorganic carbon by the C 3 mechanism (85). In Laurencia papulosa, photosynthesis is inhibited by 2 at 0.5 mM DIC (71). However, the algal extract has high PEPC and PEPCK activities. Malate pools can be detected with levels greater at night than during the day, but the fluctuation is not large enough to imply the operation of a CAM mechanism (71). Thus, this alga probably operates a C 3 ~type photosynthetic mechanism. Photosynthesis in Chondrus crlspus whole thalli, thalli pieces, and protoplasts has been examined (136, 137, 138), and all the data are in agreement. The photosynthetic rate saturates at 3-4 mM DIC, and inorganic carbon does not accumulate internally, according to measurements using the silicone oil centrifugation technique (137, 138). This alga has an external CA that functions to dehydrate HC0 3 to C0 2 and the C0 2 then passes across the plasma membrane (137, 138). Carbonic anhydrase activity is important for the function of this mechanism, as the initial carbon fixation rate decreases when CA is inhibited (137).

PAGE 34

21 The C0 2 translocation appears to be passive, since inhibitor studies do not suggest the presence of the band 3 anion transporter, a Na + /K + transporter, or any involvement of Na + in a transport mechanism (138). Therefore, internal accumulation of inorganic carbon cannot occur, as metabolic energy would have to be expended to do so. This alga can use HC0 3 in seawater via an indirect mechanism where a conversion to C0 2 by CA occurs first, and then C0 2 moves into the algal cells. Intracellular CA also plays an important role in this carbon utilization mechanism (138) Another red macroagla, Gracllarla conferta, exhibits O,insensitive photosynthetic rates and a low C0 2 compensation point under ambient DIC conditions (73). Rubisco is the major carboxylating enzyme, although PEPC and PEPCK activities are measurable. Inorganic carbon fixation occurs via the PCR cycle and supporting evidence for photosynthetic C 4 acid metabolism is lacking (73). Photosynthesis saturates at close to ambient DIC conditions and HC0 3 ~ is believed to be the main source of inorganic carbon. However, it is not known whether active uptake of HC0 3 ~ occurs or if dehydration to C0 2 occurs prior to uptake (73). In Gracllarla tenulstlpltata, an indirect HC0 3 ~ utilization mechanism based on dehydration of HC0 3 ~ to C0 2 by an external CA has been proposed (63), similar to the mechanism hypothesized for Chondrus crlspus. Carbonic anhydrase activity has been measured extracellularly and intracellularly, including an association with the chloroplast membranes (63). During photosynthesis, alkalinization of the surrounding medium depends on the Cconcentration and is inhibited by an inhibitor of external CA (63). However, the photosynthetic rate for thalli and protoplasts is higher at pH 6.5 as compared to pH 8.6, which indicates that C0 2 is the preferred C^ species. At alkaline pH, the photosynthetic rate exceeds that which could be attained based on spontaneous HC0 3 ~ dehydration alone, and when the external CA is

PAGE 35

22 inhibited, the photosynthetic rate drops to that which would be expected based on the spontaneous dehydration of HC0 3 ~ to C0 2 (63). In the presence of HC0 3 ~, Porphyrldlum purpureum has an intracellular pH of 7.3, but when the cells are carbon starved, the pH falls to 6.0. Ethoxyzolamide blocks HC0 3 ~-dependent alkalinization of the cytosol, so it is believed that CA and HC0 3 ~ are important for cytosolic pH regulation (102). As is true for any enzyme, the activity of CA is pH-dependent, so a synergistic interaction may occur between C0 2 uptake and HC0 3 transport (102). Li + also inhibits HC0 3 ~-dependent photosynthesis in Porphyrldlum. Bacillariophyta An investigation of photosynthesis in the diatom Phaeodactylum trlcornutum indicates that a HC0 3 transporter is present that depends on Na + for its operation, as Li + inhibits the HC0 3 ~-dependent photosynthetic 2 evolution (46, 102). It is hypothesized that the Na + /HC0 3 symport in air-grown and 5% C0 2 ~grown cells at the plasma membrane is driven by Na + /H + antiport (46). Carbonic anhydrase plays an important role in converting the HC0 3 ~ transported into the cells into C0 2 for use by Rubisco (46, 102). Inhibition of CA by AZ at pH 8.0 increases the apparent K Q 5 (C0 2 ) 2.5-fold, but inhibition by EZ increases the K 05 (CO 2 ) 10-fold (46). Summary The marine algae display a range of photosynthetic characteristics. Some have very C 3 -like gas exchange features in having photosynthesis that is inhibited by 2 high C0 2 compensation points, and photorespiration. Others display C^-like gas exchange characteristics, where the photosynthetic rate is not inhibited by 2 the CQ 2 compensation points are low, and photorespiration is low. Despite these features, in all algae examined thus far, the primary carboxylating enzyme is Rubisco, which points to the operation of a C,like photosynthetic mechanism. The C^-like gas exchange features are

PAGE 36

2 3 accounted for by inorganic carbon concentrating mechanisms that involve either HC0 3 uptake or a C0 2 pump. The net result is to diminish oxygenation by Rubisco, which results in the appearance of C,-like photosynthesis. However, Udotea flabellum is unique in that substantial evidence exists that supports the operation of c^-like biochemistry based on the high activity of PEPCK found in this alga, and this biochemistry may account for the C 4 -like gas exchange characteristics of this alga. Phosphoenolpyruvate Carboxvkinase Phosphoenolpyruvate carboxykinase was discovered in chicken liver in 1953 by Utter and Kurakaski and later found to be present in all forms of liver and kidney tissue, as well as mammary, muscle, and adipose tissue (152). Utter also worked with pyruvate carboxylase, and between these two enzymes, the mechanistic basis for the entry of carbon into gluconeogenesis was established (69), as these enzymes function to bypass the step in glycolysis catalyzed by pyruvate kinase (69). In addition, this enzyme is present in a variety of other organisms: plants, algae, bacteria, fungi, yeasts, flatworms, roundworms, insects, and molluscs (152). In most organisms, PEPCK catalyzes the synthesis of PEP from OAA as part of a pathway to synthesize carbohydrates according to the following reaction: OAA + ATP <===> PEP + CO, + ADP + P. with Mn 2+ 2 as a cof actor (E.C. 4.1.1.49). Depending on the organism, other nucleotides that participate in the reaction include GTP (E.C. 4.1.1.32) or ITP. The reaction is readily reversible, but in general, the decarboxylation reaction is two to eight times faster than the carboxylation reaction (152). An exchange reaction between OAA and C0 2 is also catalyzed by PEPCK and proceeds about 30 times more rapidly than the carboxylation reaction (152). The Mn 2+ ions have two roles in catalysis: the ion binds directly to the enzyme in a one to one ratio and facilitates the

PAGE 37

24 binding of PEP (152); it also forms a complex with the nucleotide species, and the complex is the catalytically active form (31, 33, 116). Algae The green macroalga Udotea flabellum has PEPCK carboxylation activity that equals the Rubisco activity (123). In this alga, PEPCK functions in the operation of a C 4 ~like photosynthetic system that reduces photorespiration. Malate and aspartate appear as early photosynthetic products that turn over within one minute, and low amounts of radioactive label are present in photorespiratory compounds (123). This PEPCK enzyme is inhibited by incubation with MPA, and as a result, less malate and aspartate are produced as early photosynthetic products, and more photorespiratory intermediates are detected (122). Thus, the C^-like photosynthetic system and decreased photorespiration are linked to PEPCK activity hypothesized to be located in the cytosol (122) The brown alga Laminaria hyperborea has PEPCK that uses ATP/ADP in catalysis. The enzyme functions for carboxylation in an anapleurotic role for replenishing the Krebs cycle to enhance growth in young fronds before light intensities are high enough for net photosynthesis to occur (159). The enzyme in Ascophyllum nodosum has a molecular weight of 60 kD and a pH optimum for carboxylation of 7.9 (84). In this species, PEPCK catalyzes light-independent carbon fixation with C0 2 as the substrate (78) The unicellular alga Euglena gracilis has high PEPCK activity when grown autotrophically with C0 2 in the light. Higher activity is present when an additional carbon source, such as glucose, is supplied to the cells and no PEPCK activity is detectable in cells grown in C0 2 in the dark (117). However, the highest activity occurs when the cells are grown in the dark with both C0 2 and glucose. When Euglena is grown heterotrophically with lactate as the carbon source, PEPCK in the cytosol functions in gluconeogenesis to provide glucose for the cells

PAGE 38

25 (117). PEPCK in this Euglena strain is suited to anaerobic C0 2 fixation and is suggested to be the key enzyme for the methylmalonyl CoA pathway for odd-numbered fatty acids and alcohols, where propionyl CoA is incorporated into wax monoesters in fermenting Euglena (117). The enzyme is activated by a combination of Mn 2+ and Mg unlike other PEPCK enzymes. This PEPCK uses GTP/GDP for catalysis, and free sulfhydryl groups of the enzyme are essential for activity (117). The marine diatom Skeletonema costatum has PEPCK that uses ADP that functions in (3-carboxylation which is superimposed on the C 3 photosynthetic pathway (104). Turnover of C, acids does not occur and the Rubisco activity is always greater than that of PEPCK. Two pools of PEPCK may exist in this diatom: one pool is always activated, allowing for carboxylation in the light, while the second pool is inactivated in the light but activated in the dark. For this dark carboxylation, a substrate synthesized in the light, perhaps a polyol, is used as a source for PEP, thereby involving polyols in the primary metabolism of diatoms (104) The diatom Phaeodactylum tricornutum has high PEPCK activity which has an absolute requirement for ADP and Mn (72). The enzyme has a molecular weight of about 70 kD as determined by gel filtration and a pH optimum for carboxylation of 6.2 (72). This enzyme catalyzes carboxylation in vivo and uses HCO," as the inorganic carbon source. The carboxylation reaction may provide an additional source of ATP for the cells (72) Higher Plants In terrestrial plants, one subgroup of those with C, photosynthesis has high PEPCK activity located in the bundle sheath cells, where the enzyme functions in the cytosol as a decarboxylase (156). In this subgroup, the PEPCK activity is 40 to 50 times greater than that in the other C, subgroups or the C, species (67).

PAGE 39

26 Three well-studied species with this metabolism include Chloris guyana, Urochloa panicoides, and Panlcum maximum (33). The PEPCK is a hexamer of 64 kD subunits with a total molecular weight of 380 kD and is active as a decarboxylase in the pH range from 7.4 to 8.2 (33). At pH 8.0, decarboxylation proceeds ten times faster than carboxylation in C. guyana (67). Although ATP/ADP is used in catalysis, the enzyme has a wide nucleotide specificity (33). The inorganic carbon species used as a substrate for the U. panicoides enzyme is C0 2 (74). Decarboxylation activity of this PEPCK is inhibited by 3-PGA, fructose-6-P (F6P), fructose-l,6-bisphosphate (F-1,6-BP) and excess ATP but stimulated by CI (33). Decarboxylation is also inhibited 40% by 0.5 mM C0 2 (74). Actually, C0 2 and HC0 3 inhibit PEPCK decarboxylation activity to nearly the same extent; this inhibition may be involved in dark regulation of the enzyme (74) PEPCK activity in Chloris guyana has no pyruvate kinase activity associated with it (67). In contrast to PEPCK from chicken liver, this enzyme does not require the presence of a thiol-reducing compound for maximal activity. However, as for all other PEPCK enzymes, Mn 2+ is essential for activity, and this ion cannot be replaced by any other divalent metal cation (33, 67, 151). The Mn 2+ binds to the enzyme at its own site, and it also binds with the ATP as MnATP 2, as this species is the actual substrate for the enzyme (33). In the presence of Mn 2+ Mg 2+ inhibits the reaction (33). In Spartlna anglica, the levels of pyruvate, alanine, and 3-PGA cannot account for all the PEP produced by PEPCK in the C 4 cycle (134). Either PEP moves directly from the bundle sheath cells to the mesophyll cells or more than one pathway of PEP metabolism is involved in this C 4 cycle (134). The reaction mechanism for PEPCK carboxylation in Panlcum maximum is fully ordered with ADP binding to the enzyme first, followed by PEP and then CQ 2 The OAA is released prior to ATP (2). However, the

PAGE 40

27 enzyme from C. guy ana has a fully random kinetic mechanism where C0 2 ADP, and PEP can bind in any order (2). Because C0 2 can bind before the other substrates, the enzyme must have a C0 2 binding site. In the C^ species Urochloa panicoides, two decarboxylation systems are present in the bundle sheath cells. NAD malic enzyme decarboxylates malate in the mitochondria while PEPCK decarboxylates OAA in the cytosol (34, 66). Studies with radioisotopes indicate that PEP comes from aspartate via OAA while pyruvate comes from malate; 2-oxoglutarate is required in this system, presumably for OAA formation (34). The major source of ATP for PEPCK activity is phosphorylation in the mitochondria rather than photophosphorylation in the chloroplasts. For this system to work, malate has to be oxidized by NAD malic enzyme at one-third the rate that PEPCK operates to produce one NADH per three ATP utilized (34). In the bundle sheath cells, mitochondrial electron transport contributes little to NADH oxidation; rather, NADH is oxidized primarily by reducing OAA to malate (66). In these mitochondria, Krebs cycle activity is not enhanced. There is PEPCK in plants other than grasses. In apple, for example, PEPCK activity peaks at the pre-climacteric stage and shortly after the fall of the petals; its role is to metabolize organic acids (17). In other species with C, photosynthesis, such as orange, lemon, avocado, and grape, PEPCK functions in the fruits to fix C0 2 in the dark (17). The carboxylation and decarboxylation reactions of Panicum PEPCK are inhibited completely by 100 pM MPA while the exchange reaction is inhibited to a lesser extent (120). The inhibitor MPA at 100 uK concentration did not affect the activity of Rubisco or NAD-malate dehydrogenase (MDH) and NAD and NADP malic enzyme are only partially inhibited (120). The activity of PEPC is stimulated 70% by 1 mM MPA (120). Thus, in Panicum, MPA substantially inhibits only PEPCK. However, in Spartlna, MPA is not a specific inhibitor of PEPCK because

PAGE 41

28 HC0 3 -dependent 2 evolution by bundle sheath strands was inhibited at pH 6.0, but not at pH 8.0 by MPA (134). Fungi Yeast In yeast, Saccharomyces cerevlslae, PEPCK is a tetramer (152) with one binding site per subunit (50) that utilizes the ATP/ADP nucleotides for reaction. The optimum pH for enzyme stability and catalysis is 6.5 (147). The molecular weight per subunit is about 64 kD with a holoenzyme molecular weight of approximately 261 kD (147). The enzyme functions in gluconeogenesis, but if a glucose source is available to the yeast, catabolite repression occurs, where PEPCK is degraded more rapidly in the presence of the glucose because the enzyme is not needed to make more glucose (106, 160). This catabolite repression is in contrast to Euglena, where PEPCK is synthesized in the presence of glucose (117). Gluconeogenesis must occur in a respiring yeast cell, as the cytochrome system and protein synthesis in mitochondria are necessary for the pathway to function (160). The catalytic sites on the enzyme contain active thiols, and each subunit has 10 cysteines that must be in the reduced form for the enzyme to be active (36). There is a Mn 2+ binding site in addition to the site where ATP/ADP-Mn 2+ binds. When ATP/ADP-Mn 2+ is bound to the active site, it affords protection against inactivation of the essential arginine residues of the site (36, 95). Anaerobe of rumens An anaerobic fungus of rumens, Neocallimastlx frontalis, has a PEPCK enzyme that is 608 amino acids long (125). The catalytic regions, with the sulfide binding site and and nucleotide binding domain, are highly conserved among fungal organisms and animals; however, the amino acid seguence of the yeast enzyme bears no similarity to the seguence of the rat, chicken, or fruit fly (125). Thus, although the enzymes may catalyze similar reactions, the proteins themselves have various

PAGE 42

29 structures. It should be noted that the yeast enzyme utilizes ATP/ADP while the other enzymes mentioned above use GTP/GDP. Bacteria In Veillonella parvula, an anaerobic bacterium of the human oral cavity, PEPCK functions in gluconeogenesis as a roundabout way of synthesizing PEP during lactate metabolism (38). The photosynthetic bacterium, Rhodo spirillum rubrum, has PEPCK that operates as a carboxylase with C0 2 as substrate (44). Bacteroides fragilis, a bacterium of the gut, has PEPCK that acts when low C0 2 concentrations are present. It refixes the C0 2 released from the decarboxylation of succinate to propionate to allow more ATP to be synthesized during glucose catabolism (37). Protozoa In Trypanosoma cruzi, the organism that causes sleeping sickness in humans, the PEPCK is located in membrane-bound glycosomes. The enzyme has a molecular weight of 84 kD and is composed of two subunits containing cysteine residues that are important for catalytic activity (150). The primary function of the enzyme is thought to facilitate the reoxidation of NADH from glycolysis by producing OAA that can be reduced to malate, and it also is involved in producing succinate, an end product of glucose metabolism (110). In addition, it functions to facilitate the complete oxidation of the amino acid skeletons that enter the Krebs cycle and operates in biosynthesis, functioning as a decarboxylase (150). Unlike PEPCK in yeast, this enzyme is not subject to repression by glucose (150). In Leishmania mexicana, a blood fluke, PEPCK is also located in glycosomes, which are unique to the trypanosomatids (105). This enzyme catalyzes C0 2 fixation and operates at a key branchpoint in the metabolism of Leishmania with PEP. High activity of MDH when PEPCK activity is also high, in the amastigote stage, may work to bring about succinate production in conjunction with fumarate reductase (105).

PAGE 43

30 Platvhelminthes The filiarial worm Setaria dlgitata, a facultative anaerobe, uses PEPCK to fix C0 2 and ferment lactate (8). This enzyme utilizes ATP/ADP for catalysis. The PEPCK in the liver fluke Fasclola hepatica operates for reverse gluconeogenesis at the branchpoint of PEP to control the ratio of fermentation products by competing with pyruvate kinase for PEP (88). Catalysis by PEPCK results in the production of acetate and propionate, while pyruvate kinase activity leads to lactate production (88). Thus, the PEPCK functions as a carboxylase in an anaerobic environment using IDP (88). The enzyme is evenly distributed between the cytosol and the mitochondria, has an optimum pH range of 5.7 to 6.7, and has an absolute requirement for Mn 2+ for catalysis. The activity of this PEPCK appears to be modulated only by the reaction substrate and product concentrations, and MPA is a potent inhibitor of catalysis (88). After OAA is produced by PEPCK, it is rapidly converted to malate by an active malate dehydrogenase, and the malate moves to the mitochondria (88). Nematodes In the helminth Ascarls suum, PEPCK functions as a glycolytic C0 2 ~ fixing enzyme and is the point at which the metabolisms of this parasite and its host diverge (116). This enzyme is functional as a monomer with a molecular weight of about 80 kD and utilizes GTP/GDP for catalysis (116). As for other PEPCK proteins, Mn 2+ binds directly to the enzyme to activate it. The active nucleotide complex can be formed with either Mn 2+ or Mg 2+ (116) In the nematode Trichlnella spiralis, PEPCK functions as a carboxylase in the fumarate reductase pathway for the production of nvalerate, acetate, and propionate (18). This enzyme uses ITP/IDP with an optimum pH of 6.6, and 98% of the enzyme is located in the cytosol with the other 2% in the mitochondria (18).

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31 Vertebrates Many of the investigations on PEPCK have been performed with chicken and rat tissue. In animals, PEPCK functions in liver and kidney tissue for gluconeogenesis, and since it catalyzes the first committed step of the pathway, it is also the rate-determining enzyme for the metabolic seguence that produces glucose from OAA (69). In gluconeogenesis, PEPCK and pyruvate carboxylase by-pass the glycolytic step catalyzed by pyruvate kinase. Within the livers and kidneys of humans, cows, sheep, cats, dogs, and guinea pigs, PEPCK is evenly distributed between the cytosol and mitochondria. In rats, mice, and hamsters, a larger proportion of PEPCK activity is found in the cytosol. In the livers of chickens, pigeons, and rabbits, more PEPCK activity is found in the mitochondria, but the distribution in the kidney is fairly even between the mitochondria and the cytosol (69). Chickens This PEPCK is a monomer with a molecular weight of 70 kD, composed of 622 amino acids (43). The nucleotide used in the reaction is GTP/GDP (130). In the mitochondrial enzyme from embryo liver, the enzyme is activated by micromolar concentrations of Mn 2+ in the presence of millimolar concentrations of Mg 2+ (131). The OAA-C0 2 exchange reaction is activated synergistically by Mn 2+ and Mg 2+ (131). The regulation of PEPCK is different in birds and mammals in that avian livers are unable to synthesize glucose from pyruvate or amino acids (69). However, chickens have a high glucose concentration in the blood, and avian kidney tubules are able to use both pyruvate and lactate. In liver, lactate is the major precursor for glucose synthesis (69). Gluconeogenesis from pyruvate and amino acids requires transfer of reducing eguivalents and carbon from the mitochondria to the cytosol. In tissue with a cytoplasmic PEPCK, malate from the mitochondria moves to the cytosol, where it is oxidized by MDH to OAA, thereby producing NADH (69).

PAGE 45

32 In contrast, in tissues that have very low cytosolic PEPCK activity, such as chicken liver, the cytosolic NADH arises from the transformation of lactate to pyruvate in the cytosol. The low level of cytosolic PEPCK activity in chicken liver cells is attributed to posttranslational regulation, as considerable levels of PEPCK mRNA are present (157). Thus, the cytosolic enzyme in the kidney in a starvation state uses gluconeogenesis to convert the amino acids resulting from protein degradation into glucose (69). During starvation, the mRNA level for the cytosolic enzyme increases, after which PEPCK is synthesized (157). The mitochondrial PEPCK functions as a part of the Cori cycle to oxidize lactate to pyruvate (69). The cytosolic and mitochondrial enzymes are synthesized from two different mRNA molecules (43), they exhibit subtle differences in ligand interactions at the active site, the proteins have different molecular weights, and they do not crossreact to antibodies produced from the other form of the enzyme (69). Another difference between the two enzymes is that the cytoplasmic form catalyzes the OAA-C0 2 exchange reaction at a rate 30-100% greater than that catalyzed by the mitochondrial enzyme (130). The mitochondrial enzyme is synthesized in the cytoplasm as a precursor, having a signal peptide that is later excised after its arrival in the mitochondrion (69). In fact, the cytosolic and mitochondrial forms from one species share only 56% homology in their amino acid seguences, while there are many similarities between the cytoplasmic enzyme from species from different classes, such as the chicken and rat (43, 69). Synthesis of the mitochondrial PEPCK is constitutive (43), while synthesis of the cytosolic enzyme increases in response to epinephrine, glucocorticoids, thyroid hormones, and glucagon which acts via cAMP (43, 69). Insulin inhibits cytosolic PEPCK synthesis. In chicken liver cells, PEPCK is also found in the nucleus (145). The enzyme in this location functions for anapleurosis, in the nuclear

PAGE 46

33 synthesis of N-acetyl-neuraminic acid, and the synthesis of glycosylated high mobility group proteins, which are non-histone proteins associated with the nuclear protein matrix (145). Rats In rat liver cytosol, PEPCK functions in gluconeogenesis (149) and is a protein of 621 amino acids with a molecular weight of approximately 70 kD (9, 31). The enzyme utilizes GTP/GDP and is activated by micromolar concentrations of Mn 2+ (31). The mRNA is about 2600 nucleotides long, and a portion of the gene favors Z-DNA formation (9). The hormone regulation of gluconeogenesis in rats and chickens is similar. Glucagon via cAMP and glucocorticoids stimulate PEPCK activity while insulin inhibits it (9). These hormones exert their effects by altering enzyme synthesis via mRNA amounts and translation (9). In rats that were starved and re-fed, serotonin increases the level of mRNA coding for the cytosolic form of PEPCK in the kidney, liver, and small intestine. The level is higher than found in the same tissues after 24 hours of starvation, and the serotonin stimulates cAMP levels within one minute of treatment (162), thus further affirming that production of PEPCK occurs in response to cAMP levels. Other Mammals In camels, PEPCK functions in gluconeogenesis to maintain the high level of blood glucose found in these animals (1). More enzyme occurs in the kidney relative to the liver, but both isozymes have a molecular weight of 80 kD and pH optimum for activity of 7.2. Rabbit PEPCK in the liver occurs in both the mitochondria and cytosol. The molecular weight of the cytosolic form is 68 kD, while that of the mitochondrial form is 5 6 kD as determined by gel filtration (58). The cytoplasmic form functions in gluconeogenesis from amino acids, but the function of the mitochondrial enzyme is unclear. It may be involved in remobilizing lactate as a part of the Cori cycle (58).

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34 Differences in molecular weight and function suggest differences in the amino acid seguence between the two forms (58). In mouse pancreatic islets, the PEPCK uses ITP/IDP for catalysis and is activated by Mn 2+ (68). Preincubation of the enzyme with ATPMg" inhibits the Mn 2+ -stimulated activity over time. Two fishes have been examined for their PEPCK activities. In the carp hepatopancreas, one form of PEPCK is present that has a molecular weight of 83 kD as determined by SDS electrophoresis and a pH optimum of 7.7 for decarboxylation and 7.2 for carboxylation (87). The liver of the rainbow trout has isozymes with molecular weights and pH optima similar to the carp enzyme (87). The enzymes function in gluconeogenesis. Of metal ions, Mn 2+ is the best activator (87). Summary The PEPCK enzymes that utilize ADP/ATP for catalysis are generally either tetramers, as in yeast, or hexamers, as in the higher plants. The subunits typically have molecular weights in the 65 kD range. In contrast, the enzymes that use GDP/GTP for catalysis are monomers that have molecular weights in the 70 to 80 kD range. In most organisms where PEPCK is found, the most common location of the enzyme is in the cytosol, and the optimum pH for catalysis ranges from 6.2 to 7.9 among the organisms. For its reactions, PEPCK is Mn 2+ -specif ic. The ion binds to a site directly on the enzyme and may also form a nucleotide complex for the reactions. In organisms where PEPCK functions as a carboxylase, C0 2 is the inorganic carbon substrate for the enzyme. In the algae, PEPCK functions as a carboxylase for anapleurosis and synthesis of fatty acids. In the higher plants, the enzyme operates as a decarboxylase in one of the C^ photosynthetic subgroups. In the vertebrates, PEPCK is a decarboxylase in the gluconeogenic pathway. Thus, this enzyme catalyzes a range of reactions for metabolism, and its activity in both directions is inhibited by MPA.

PAGE 48

35 In Udotea, where PEPCK operates presumably in the cytosol, the enzyme is hypothesized to function as an oligomer that uses ADP/ATP for its reactions. The Mn 2+ ion also appears to be important for the reactions to proceed. This PEPCK activity appears to be crucial for the operation of the C 4 ~like photosynthetic mechanism, as its inhibition with MPA shifts the photosynthetic metabolism to Cj-like. Thus, Udotea PEPCK appears to share some features with PEPCK enzymes from some algae, yeast, and the higher plants. One of the purposes of this investigation was to learn more about the characteristics of PEPCK in Udotea to understand better how it plays a role in the C 4 -like photosynthetic mechanism of this alga.

PAGE 49

CHAPTER 2 INORGANIC CARBON UTILIZATION IN THE MARINE MACROALGA UDOTEA Introduction Udotea, a marine siphonaceous macroalga of the Chlorophyta, has been found to exhibit C^-like photosynthetic characteristics, but the mechanism which produces these features requires more adequate investigation. C^-like gas exchange properties, such as Oj-insensitive photosynthesis, low C0 2 compensation point, and low photorespiration rate can be attributed to one of two general mechanisms: HC0 3 ~ utilization or C 4 acid metabolism. It is evident that C 4 acids are an important part of the photosynthetic metabolism of Udotea, as high activities of enzymes typically associated with this type of metabolism are found in this alga (123). In addition, during light fixation of C0 2 there is an early appearance of C^ acids which turn over rapidly, suggesting that these acids are not just metabolic end-products (123). The purpose of this investigation was to determine if Udotea also uses HC0 3 ~ as a part of its photosynthetic carbon assimilation mechanism. Bicarbonate utilization could occur via an indirect mechanism, in which an external CA converts HC0 3 ~ from the seawater into C0 2 that either diffuses across the plasma membrane as proposed for the red alga Chondrus crlspus (138) or be actively transported across, as reported for the green microalga Chlorella (126). Alternatively, could be used directly in a system where the ion is transported across the plasma membrane on a carrier which may or may not require energy to operate (135) Since Udotea is a calcareous alga, the role of calcification during inorganic carbon asssimilation must also be considered, as 36

PAGE 50

37 calcification and photosynthesis are closely linked in other calcifying organisms (24). The results presented in this paper suggest that C0 2 is the primary species of inorganic carbon used in photosynthesis, but some energy-dependent HC0 3 ~ use also occurs. A possible model for calcification is also considered. Materials and Methods Materials Specimens of the marine macroalga Udotea conglutlnata (Ellis and Solander) Lamouroux (division Chlorophyta) were collected with intact holdfasts in late fall, spring, and summer at the mouth of the Crystal River in the Gulf of Mexico, Florida, in water ranging from 1.0-1.5 m in depth. After collection, the plants were washed clean of epiphytes and transplanted into 20 L aguaria with mollusc shell fragments on the bottom, filled with filtered, aerated, natural seawater. The aguaria were placed into a growth chamber under a 2 5C/12-h photoperiod with a guantum irradiance of 5-10 jumol rrf 2 s" 1 (400-700 nm) and a 23C scotoperiod. The seawater was changed every three to four weeks. Algae used for the investigations appeared healthy and had lush green color. Gas Exchange Experiments Net photosynthetic rates were determined as 2 evolution with a Hansatech electrode system at 25C and 21% and 2% gas phase O, (equivalent to 220 and 22 pM 2 respectively) in synthetic seawater buffered with 10 mM Tris-HCl, pH 7.7, 10 mM Tris-HCl, pH 8.2, or 10 mM CHES-NaOH, pH 9.0, containing various amounts of dissolved inorganic carbon, added as NaHC0 3 The artificial seawater was prepared according to Drechsler and Beer (47) and contained 450 mM NaCl, 10 mM KC1, 10 mM CaCl 2 and 30 mM MgS0 4 The salinity of this solution was determined by the HW Harvey method (96). A 10 mL sample of water to which four drops of 0.41 M K 2 Cr0 4 had been added was titrated with 0.16 M AgN0 3 solution until a brick-red color persisted with stirring. The salinity, in parts

PAGE 51

38 per thousand, was equivalent to the volume of AgN0 3 solution needed to titrate the 10 mL sample. The quantum irradiance during the photosynthetic measurements was 300 pmol nf 2 s" 1 (400-700 nm) The rate of C0 2 production in the photosynthetic medium arising from hydration and dehydration of the various inorganic carbon species present in seawater was calculated according to equation 6 from Johnson (76) using the constants determined for seawater with a salinity of 34 parts per thousand at a temperature of 2 5C. The calculation for the hydration rate of C0 2 in seawater is presented according to the method of Johnson (76). 5(C0 2 )/6t = -(K CQ2 + K QH K w /a H )(C0 2 ) = (k d a H + K, HC03-) ( HC0 3 M = mol L 1 For seawater at 25C and 34 parts per thousand salinity: K CQ2 = 0.0362 s" 1 K 0H K w = 13 4 x 10 11 m l L~ 1 s" 1 K d = 3.52 x 10 4 L mol" 1 s" 1 K HC03= 1 11 x 10" 4 s" 1 a H = [H + ] At pH 8.2 and 2 mM DIC: [H + ] = 6.31 x 10" 9 M [C0 2 ] = 0.00601(DIC) = 1.20 x 10~ 5 M [HC0 3 ] = 0.898(D1C) = 0.001796 M Therefore, substituting into the equation: 5(C0 2 )/St = -[0.0362 + (13.4 x 10~ 11 ) /6 31 x 10" 9 ](1.20 x 10" 5 ) + [(3.52 x 10 4 )(6.31 x 10 -9 ) + 1.17 x 10" 4 ) ] (0 001796 ) 5(C0 2 )/5t -[0.0362 + 0.0212] (1.20 x 10 -5 ) = (3.39 x 10 -4 ) ( 001796 ) = -(0.0574) (1.20 x 10" 5 ) + 6.09 x 7 = -(6.89 x 10~ 7 ) + (6.09 x 10 -7 ) = -8 x 10 -8 s" 1 x M = -0.08 x 10~ 6 s" 1 x M = -4.8 x 10~ 6 min -1 x M

PAGE 52

39 The effect of CaCl 2 on photosynthesis was measured in artificial seawater prepared with and without CaCl 2 Sections of thallus, about 15 mm by 15 mm, from the tip of the thalli, were incubated for 15 min in unbuffered, artificial seawater with or without 10 mM CaCl 2 at a quantum irradiance of 150 jumol m" 2 s" 1 (400-700 nm) The photosynthetic rate measurement in the presence of 2 or 5 mM DIC commenced with adding the alga to the oxygen electrode chamber, which already contained 2 mL artificial seawater buffered with 10 mM Tris-HCl, pH 8.2. Photosynthesis was measured for 15 min. An aliquot of the reaction mixture in the 2 electrode chamber was removed to measure the initial total inorganic carbon. A 100 pL sample was injected into 3 mL 0.1 N H 3 P 4 bubbled with N 2 in a flask, and the C0 2 released was measured with an infra-red gas analyzer and recorded on an integrator against a known concentration of a KHC0 3 standard. Another aliquot of the reaction solution was removed after the photosynthesis measurement to record the total DIC remaining. The influence of several potential effectors on photosynthesis was examined, including 40 mM KI, 0.24 mM DIDS, and 0.1 mM vanadate. The vanadate solutions were prepared according to three different methods. In the first method, solution A, 5 mM Na 3 VO A was dissolved in 20 mM NaOH and allowed to stand overnight at room temperature (57). In the second method, solution B, Na 3 V0 4 was dissolved in deionized water (20 mL) to produce a 5 mM solution and heated for 3 h at 40C (109). After cooling, the volume was readjusted to 20 mL with deionized water, and the pH was adjusted to 9.5 with MES powder. The absorbance of a 0.1 mM solution was recorded at 2 68 nm against a water blank in order to determine the true concentration of the solution. The molar extinction coefficient used was 1.44 x 10" A as determined by Cantley et al (35). The pH was then adjusted to 7.2 with MES powder and the solution was stored in a glass stoppered flask at room temperature until used. In the third method, solution C, the solution was prepared on the day of

PAGE 53

40 the experiment as 100 mM solution in 20 mL deionized water (142). The pH was adjusted to 9 5 with MES powder, and the solution was boiled until colorless. After cooling, the pH was adjusted to 7 5 with MES powder, followed by a second boiling. After cooling, the volume was adjusted to 20 mL with deionized water and the pH adjusted to 7 5 with MES powder. Potential effectors were added into the standard reaction mixture, consisting of artificial seawater buffered with 10 mM Tris-HCl at pH 8.2, to which NaHCOg was added to achieve various DIC concentrations The 2 electrode system was also used to determine which species of inorganic carbon was used for photosynthesis. These photosynthetic measurements were performed at 9C to slow the equilibration between CO., and HCO3-. The procedure that Cooper et al (44) used to determine the DIC substrate of carboxylation enzymes was modified for use on thalli pieces. The Udotea thallus section was placed into a chamber containing 2 mL artificial seawater buffered with 10 mM Tris-HCl at pH 8.2, at a quantum irradiance of 300 pmiol nf 2 s" 1 (400-700 nm) When a stable line was achieved on the chart recorder, either 5 mM NaHC0 3 or 5 mM NaHC0 3 acidified with 0.1 N HC1 was added to the chamber with a Hamilton syringe. If an acidified solution was used, the acidification was done in the syringe, and the solution was added to the reaction chamber only after bubbles of CO., appeared in the syringe. In this manner, a significantly larger proportion of either C0 2 or HCO3" was added to the reaction chamber. The photosynthetic rate was followed for a maximum of one minute. pH Drift Experiments Udotea thalli without holdfasts were placed into 60 mL glassstoppered bottles containing unbuffered, artificial seawater to which 2 mM NaHC0 3 was added (94). The pH of the starting solution was recorded and a sample was removed to measure the true concentration of total inorganic carbon present at the start of the experiment. The stoppered

PAGE 54

41 bottles were placed into a shaking water bath maintained at 32C under a quantum irradiance of 200 ^mol rrf 2 s~ 1 (400-700 nm) for 6 h. After removal from the water bath, the bottles were allowed to return to room temperature, at which time the final P H of the solution was measured. An aliquot of water was removed to measure the remaining total DIC, and the alkalinity of the water was measured by titrating a 10 mL sample of the water with 0.121 N HC1 to pH values of 4.2, 3.9, and 3.7 (94). The quantities of acid added and the exact pH values achieved were used to calculate the alkalinity of the samples. The Udotea thalli were removed and the fresh weights recorded. The thalli were then dried in a 70C oven for at least 24 h and dry weights determined. The HCO," dehydration rate was calculated using equation 6 and the constants from Johnson (76) Carbona te Salts in the Algal Skeleton Thalli that had been oven-dried were placed into Petri dishes and treated with 6 N HC1 to liberate the carbonates in the plant skeleton. After bubbling ceased, the acid was pipetted off and the remaining thalli allowed to dry. After drying, the weights were measured and compared to the fresh weights and the dry weights prior to acid treatment Carbonic Anhydrase Assay Carbonic anhydrase was assayed according to the method of Drechsler & Beer (47). At 10-12C, the time for a pH change of two units was recorded for a solution of 5.8 mL artificial seawater, 0.2 mL Tris-HCl, final concentration 10 mM, pH 8.6, to which 4 mL of ice-cold C0 2 -saturated water was added. The time required to achieve the same pH change was also noted when 10 units of carbonic anhydrase or a piece of Udotea thallus were added. The thallus had been cut the previous day so that wound healing would occur (41) and the enzyme being measured would be located on the thallus, rather than having leaked from the cytosol of the alga.

PAGE 55

42 Thallus Surface pH Udotea thalli sections cut the night before experimentation were placed into Petri dishes containing 40 mL unbuffered artificial seawater with 2 mM NaHC0 3 added. These sections were incubated in the growth chamber for 15 min, under a quantum irradiance of 5 pmol itf 2 s~ 1 after which time the thalli were moved to a fresh solution of 2 mM NaHCO-, in artificial seawater. The thalli sections were then exposed to a quantum irradiance of 300 /imol m" 2 s _1 (400-700 run) while the pH of the thallus surface was recorded with a microelectrode (49). Measurements were made at various points on the thallus, and the change in the pH of the water from the beginning of the thallus measurements until the end was also recorded. Thallus measurements were also made in the presence of 40 mM KI, 0.24 mM DIDS, and 1 mM vanadate, as well as in the dark, and for the water in the absence of thalli. Chlorophyll Determination Chlorophyll was determined by the method of Arnon (3). The thallus was ground at 4C in a mortar and pestle in 2 mL 10 mM Tris-HCl, pH 7.3. A 0.2 mL aliquot of the homogenate was pipetted into 0.8 mL 100% acetone. This solution was incubated on ice, in the dark. After 15 min, 2 mL 80% acetone was added to the solution, which was incubated for another 15 min period on ice in the dark. The solution was then centrifuged at 3600 g for 5 min at room temperature. The absorbances of the clear solution at 645 and 663 nm were determined against a blank of 80% acetone. The equation for the calculation is: [ (A663 x 0.00802) + (A645 x 0.0202)] x 5 (dilution factor) x 2 (total mL in grind) = total mg Chi in thallus. Results An examination of the structural components of the Udotea thallus indicated that the fresh weight of the thallus was about double the dry weight, and nearly 50% of the dry weight was composed of carbonate salts that could be removed by treatment with HC1 (Table 2-1).

PAGE 56

43 Table 2-1. Proportion of Udotea dry weight composed of carbonates. DW refers to the dry weight remaining after acid treatment. Carbonate Thallus Fresh Weight Dry Weight FW/DW Dry Weight After Acid FW/DW A Component of Skeleton g a % 1 0.97 0.44 2.20 0.26 3.73 41 2 1.59 0.75 2.12 0.36 4.42 52 3 1.72 0.73 2.36 0.40 4.30 45 4 1.07 0.49 2.18 0.25 4.28 49 5 1.03 0.50 2.06 0.25 4.12 50 Mean ___ 2.18 + 0.11 4.17 0.27 47 4

PAGE 57

44 The total amount of 2 evolved during photosynthesis by Udotea at 2 mM DIG in the absence of CaCl 2 was similar to that with CaCl 2 present, although with 5 mM DIC in the medium, 22% more 2 was evolved during the same time period when CaCl., was included in the photosynthetic reaction mixture (Table 2-2). However, a noticeable difference occurred with regard to the net change of the total inorganic carbon in the medium when CaCl 2 was omitted from the medium. The presence of the CaCl 2 seemed necessary for inorganic carbon to be removed from solution, as in its absence, more DIC was present at the end of the 15 min photosynthetic period than at its beginning. In the presence of CaCl 2 the Quantity of inorganic carbon removed by the thallus section in 5 mM DIC was 6-fold greater than the amount removed in 2 mM DIC. When CaCl was absent, about twice the amount of inorganic carbon was present after photosynthesis in the chamber which started with 2 mM DIC as compared to the chamber with 5 mM DIC. The species of inorganic carbon used for photosynthesis was investigated (Fig. 2-1). By using a method modified from a study of Cooper et al (44) on isolated enzymes, whole-thallus photosynthesis was carried out at 9C so as to slow the equilibration of the carbon species added to the reaction medium. The experiment was performed at pH 8.2. When Die was added as virtually all co 2 the photosynthetic rate was five-fold higher than when the DIC was added so that 90% of the carbon was present as HCO3-. When CA was added along with HC0 3 ", no stimulation of photosynthesis was observed. When CA was added with COp, the photosynthetic rate dramatically decreased by five-fold, to the level attained in the presence of HCO ~ alone. In a pH drift experiment, where intact Udotea thalli were incubated for six hours under a quantum irradiance of 200 /jmol m" 2 s _1 (400-700 nm), the final pH attained by the plants was approximately 8.3 (Table 2-3). Of the total DIC present at the conclusion of the experiment, the CQ 2 component was 5 /jM, while nearly 88% of the

PAGE 58

45 Table 2-2. Effect of 10 mM CaCl 2 on Udotea photosynthesis at pH 8.2. Time elapsed during photosynthetic measurement was 15 min. Values reported are the mean of 2 measurements SD. With Without With Without Ca Clp CaCl, CaCl, CaCl., DIC Concentration Total Q 2 evolved Net Change in total DIC "^ mmol g" 1 FW 2 0.11 0.03 0.10 0.06 -0.23 2.17 5 0.14 0.01 0.11 0.03 -1.30 1.10 Note: A positive value indicates that more inorganic carbon was present in the chamber after the photosynthesis measurement was made than at the start.

PAGE 59

46 < (J < u l-M Md i.B z O lotury re it TD -p 3 C ^^ o OP** U o H tj jes -H Q 3+1 Im re -P -h J* S ft CD -h p.tn c m c '.^ CD ij XI •H rvJsL -p •P O CD c o > id i m H rl O JJI Hi n O >i A c 2oa 3 (0 id c v id CD m !-l O 3 a -P C X O -H m •-I e -P id C 5 E-y U O -P -H -rH c C -P -P CD CD O id fiCD O (ti coo) C CD -H U U O U e w a o cd CD X >.CJ ta x: w -t M -p P Q p CO (d o H 1 o a s e -p < O -P CN to in rA id P 0) CD 3 Di h TS XI O C 3
PAGE 60

47 Table 2-3. Final pH of three replicates of Udotea following a pH drift experiment in 200 pmol quanta m 2 s 1 for 6 hours at 32C. Values reported are the mean of 3 replicates SD. Mean Final pH Total DIC Final co 2 Final HC0 3 HC0 3 ~ Dehydration Rate 8.26 0.40 1000 464 pM 5 2 877 407 /jmol min~ 0.29

PAGE 61

48 inorganic carbon was present as HC0 3 ". At this pH, C0 2 was produced from HC0 3 at the rate of 0.29 ^mol min" 1 In another pH drift experiment, where the amount of DIC present at the start of the incubation period was only that added as a result of bubbling the artificial seawater with air for 0.5 h, the final pH reached was 8.95 0.42 for 15 samples. The manner in which pH affects photosynthesis was investigated (Fig. 2-2). For similar CC> 2 concentrations, the additional HCO~ available at higher pH values was able to increase photosynthesis. At pH 9.0, 0.06% of the total DIC is C0 2 and 59.6% is HC0 3 ~, at pH 8.2, 0.6% is C0 2 and 89.8% HCO3", and at pH 7.7, 2.0% is C0 2 and 94.8% HCOj". This higher photosynthetic rate suggested that some HC0 3 ~ was being employed for photosynthesis in addition to the C0 2 although the preferred species was C0 2 (Fig. 2-1). It can also be seen that even though 30 mM DIC was added at pH 8.2 and 50 mM DIC at pH 9.0, the photosynthetic rate still was not saturated due to the small proportion of inorganic carbon present as C0 2 The rate of C0 2 production from HC0 3 was calculated for this system using the equation of Johnston (47) (Table 2-4). If the rate of photosynthesis was greater than the rate of spontaneous C0 2 production from HC0 3 ", then the evidence suggests that the additional photosynthetic rate was supported by HC0 3 utilization (47). At both 1 and 2 mM DIC, the measured photosynthetic rate for Udotea was greater than the rate predicted from spontaneous HC0 3 dehydration (Table 2-4). Udotea thalli were investigated to determine whether any external CA activity was present. The time for spontaneous HC0 3 ~ dehydration was 13 s, and adding 10 units of CA decreased the time by nearly 50% (Table 2-5). However, the presence of the Udotea thallus actually slowed the rate to 19 s, thus suggesting that no external CA was present on the thallus.

PAGE 62

49 O (J i-M Mi-B e O Pwr/ o CM 00 ffi p^ 4-1 o 0) (!) Vt 3 -P X -^ s c -p a (!) u a *H K •r-l 01 0) fl 4J C Pi en O -p U .c (M ft i CN H rt 0) > M 3 j5 Cn-P -H rH &l 3

PAGE 63

50 Table 2-4. Comparison of photosynthetic rates for Udotea thallus sections measured at pH 8.2 and 300 pmol quanta irf 2 s" 1 and the predicted rates dependent upon the spontaneous rate of C0 2 production. The predicted rates were calculated according to Johnson (76). Measured photosynthetic rates are the mean of 4 replicates SD. Photosynthetic Rate DIC 0? Measured Predicted ™ % /umol 2 h~ 1 1 21 0.37 + 0.08 0.29 2 0.57 0.24 2 21 0.44 0.10 0.58 2 0.78 0.07 5 21 0.80 0.25 1.46 2 1.18 0.17

PAGE 64

51 Table 2-5. Measurement of hydration time for C0 2 at 10-12C, recorded as a pH change of 2 units. Values reported were the mean of four or five replicates SD. The net photosynthetic rate at 2 mM DIC for the thalli examined for the presence of external CA was 26.47 + 5.77 fumol 2 g" 1 FW Condition Time for Reaction sec Spontaneous 13 3 With 10 units CA 8 1 With Udotea intact thallus 19+2

PAGE 65

52 Udotea photosynthesis was examined in the presence of several potential effectors of HC0 3 use. In the presence of 40 mM KI, where the I" may interfere with HC0 3 uptake, photosynthesis was inhibited 26 to 35%, and the inhibition increased with increasing HC0,~ concentration (Fig. 2-3). These measurements were made without any pre-incubation of the thallus in KI The photosynthetic rate was also inhibited in the presence of 0.24 mM DIDS, (Fig. 2-4), which inhibits transport by the band three anion transporter in red blood cells. The inhibition ranged from 13% at 3 7 mM HC0 3 to 33% at 0.5 mM HCO, Vanadate halts ATP synthesis by interfering with phosphate binding at plasma membrane ATPases (35). Each bar in Fig. 2-5 represents four alga pieces incubated for a period of 2 h in 0.01 mM vanadate solutions prepared by different methods. Several solution preparations were used to determine whether the vanadate ion species present in a particular solution was more effective at inhibiting the ATPase, if it were present. Photosynthesis was inhibited only 3% after 5 min incubation in solution A (Fig. 2-5), but this inhibition increased to 34% inhibition after a 2 h incubation period. Incubation in solution B did not appear to inhibit photosynthesis of Udotea thalli. For thalli incubated in solution C, photosynthesis was inhibited 19% after just 5 min, but no further inhibition resulted after prolonged incubation. Surface pH measurements of udotea thalli exposed to a quatum irradiance of 300 jumol m -2 s" 1 were made on thallus pieces incubated in unbuffered, artificial seawater containing 2 mM DIC at 25 to 30C. With no additions to the medium, the thallus pH was 0.1-0.2 units lower than the surrounding water (Table 2-6). During the course of the measurements, the water pH dropped 0.1 unit in the light. In the presence of 40 mM KI, the thallus surface was up to 1.4 pH units lower than the surrounding water. Apparently, the presence of the KI stimulated H + extrusion; the water pH dropped 0.4 units in the same

PAGE 66

H O u $ O o 53 o IB w 0 o to I IH 5 E -P • O Tl (1)
PAGE 67

54 o o X o u o o o CO X (0 P tC a> 4J o t IS a> +J ca H u •H +J 0) £ en o +> o A a c o w p t -M Md U _B z O louury m t3 O 0) TS •4-1 T3 O <0 0) 4-1 W M-l Q m H Q # £ I CN CN (U • S^ O & — •H O

PAGE 68

55 U -M Mdi-B s O PLU^ CD 4-1 -P V4 c 0) 0) 3 33 H ft 0) to m C i 4-> 4-> H id d) CD TS £ > (C -rH C TS 4-> (0 C U > td d) ft 0) CO 01 j3 rH (U H n) ti -rH *4 • Q) TS -H +J 0) rH td T3 r-tS-O (C (0 £ a 4-> -H o hj T3 T3 0) (D C -u 4-i td O td t3 CJ e> -H m T3 tw C • o -^ ft; to tn tn •rH J C to O d) -rH £ 01 -P 4J TS 3 COH >i.C 01 4-> tO O 0) 4J g d) O 4-> £ -P • (1) 44 *• 4J -h (1) a cd Tl .C -— TS -P td d) a) c d) £ tn (S 54 4-) • > £ -rH r-P s 4-1 n O O 0) -P V4 -P 3 •O Di-P .— d) C Xi 44 -r4 -rH "— r 4-1 -a S H M ID o a 00 O • O -rH tID td 4-1 1 o oj ts cd *-* d) d) HJ (U *4 iH — s-i id 3 ft.fi ro Oi 0) o 0> -rH ^ Id • fa ft (1) r-

PAGE 69

56 T f2 ble li 2-6 Surface pH measurements of Udotea thalli under 300 pmol m s irradiation (400-700 nm) except as noted, at 25C. The incubation medium for each was artificial seawater with 2 mM DIC added. ^ The two pH values reported for the water were at the beginning and end of the experiment. Each set of values reported was for eight to ten measurements on one piece of thallus. The tip values were recorded at the natural apical end of the thalli. Conditions Water Thallus Tip Thallus Center pH No additions 8.73 8.32 0.06 8.21 0.08 8.61 8.47 0.06 8.58 0.10 8.47 0.09 8.55 0.06 + 40 mM KI 8.59 8.34 0.09 7.18 0.68 8.22 7.42 0.16 6.99 0.30 + 0.24 mM DIDS 8.79 8.05 1 0.83 8.31 0.08 8.64 8.27 0.13 8.29 0.10 + 1 mM Vanadate 7.54 7.61 0.14 7.44 0.03 V.48 7.23 0.12 7.42 0.02 No plant 8.90 8.77 Dark 8.80 8.43+0.09 8.39 0.09 8.84 8.41 0.19 8.42 0.04 Dark, No plant 8.92 8.90

PAGE 70

57 period. In the presence of 0.24 mM DIDS, the thallus surface was 0.40.6 pH units lower than the water, and the water also dropped 0.2 units during the readings. The presence of 1 mM vanadate lowered the water pH; however, the pH of the water did not change during the course of the measurements, and the thallus pH remained similar to that of the water. When no thallus was present in the water in the light, the pH dropped by about 0.1 unit over a 15 min period. In the dark, without a thallus piece, the water pH remained essentially unchanged. When a piece of thallus was incubated and measured in the dark, the pH of the water remained about the same, while the pH of the thallus was 0.4 pH units lower than the surrounding water, thus indicating that the reduced pH of the Udotea thallus surface, perhaps caused by H + extrusion, was not dependent on light or photosynthesis. The effect of £ on photosynthesis at pH 8.2 was examined for the Udotea thalli (Table 2-7). The photosynthetic rate was inhibited 35 to 40% by 21% 2 as compared to 1% 2 in the gas phase. However, increasing the DIC had no effect on the degree of 2 inhibition of photosynthesis, thus suggesting that the 2 inhibition was not a photorespiratory phenomenon. Discussion The photosynthetic studies of Udotea thalli indicated that although C0 2 was used more readily for photosynthesis, there appeared to be some capacity for using HCOj". The C0 2 could enter Udotea siphons by diffusing across the plasma membrane or it could be transported across, as hypothesized for the marine macroalga Stichococcus baclllarls (102), the green microalgae Chlorella ellipsoldea (127) and C. saccharophlla (126), and the cyanobacterium Synechococcus sp. (7, 52). The C0 2 pump in the colonial green alga Scenedesmus obllquus, grown on high CC> 2 and then adapted to air levels of C0 2 is accompanied by plasma membrane CA formation (143). Other species in the Chlorophyta, such as Ulva lactuca and U. fasciata, appear to take up HC0 3 directly (6, 94), possibly by a

PAGE 71

58 Table 2-7. Effect of oxygen concentration on photosynthesis in Udotea at pH 8.2 and 300 ^mol quanta m d s n Values reported are the mean of 4 replicates + SD. DIC C0 2 HC0 3 2 Photosynthetic O 2 Rate Inhibition mM ^M mM % ^mol Og g" 1 % FW h -r 1 9-84 0.93 21 4.85 1.62 2 7.80 + 4.19 38 2 19.68 1.85 21 6.23 0.55 2 10.43 1.42 40 5 49.20 4.64 21 10.40 2.02 2 16.11 4.22 35

PAGE 72

59 HC0 3 "/OH" antiport system (12). In the green unicell Chlamydomonas relnhardtii, cells adapted to air levels of C0 2 after growth at 5% CO ? reportedly utilize HC0 3 in a DIC concentrating mechanism (39, 103). This mechanism also includes an appearance of external CA activity. Thus, CA may function as a component of either C0 2 or HC0 3 ~ utilization systems. Carbon dioxide use in Udotea would have to be by diffusion or a C0 2 pump that does not reguire external CA activity, as external CA activity was not measurable on Udotea thalli. Bicarbonate use in two of the Ulva species is hypothesized to occur by a HC0 3 ~/OJT antiport, but the pH of the Udotea thalli did not support the occurrence of OH" extrusion. External CA activity is also a component of some HC0~ utilization mechanisms, but this activity was lacking in Udotea. Thus, DIC utilization in this alga probably does not occur via any of these mechanisms. The experiment modified from Cooper et al (44) of investigating which species of DIC was used for photosynthesis indicated that C0 2 use resulted in the most rapid photosynthetic rate. This experiment was predicated on a lack of external CA on the Udotea thallus. The experiment to determine the inorganic carbon substrate for photosynthesis was performed at pH 8.2 at 9C to slow the eguilibration of the various carbon species. The reduced temperature also slowed the photosynthetic rate. When the inorganic carbon was added as largely HC0 3 even adding exogenous CA to the system did not result in a rapid enough production of C0 2 to raise the photosynthetic rate above that achieved when HCOj" alone was present. Carbonic anhydrase facilitates the rapid establishment of the equilibrium among the various carbon species for a given pH. When most of the DIC was added as C0 2 adding CA resulted in a rapid shift of most of the inorganic carbon to HC0 3 ", the species that predominates at pH 8.2, and this reduced the photosynthetic rate substantially. Thus, it appeared that the

PAGE 73

60 photosynthetic rate dependended on the low COp concentration, as Fig. 22 indicated that 0.03 mM CO., could support the same rate observed in this experiment. If little or no HCO," use occurred, then the low concentration of C0 2 present in seawater in equilibrium with air (10 uM.) is a major factor for Udotea photosynthesis in the environment. The alga must have a mechanism other than HCO," utilization to elevate the internal carbon concentration above that in the surrounding water. Although in a number of species of red, brown, and green microand macroalgae, external CA activity has been found (15, 63, 138, 141), Giordano and Maberly (61), in a survey of 34 species of macroalgae, were unable to find any correlation between the presence of CA activity and the relative ability to use HCO, for photosynthesis, indicating that external CA activity is not always essential for HC0 3 ~ utilization. However, CA activity does play a role in some HCO," use mechanisms, including that in the red alga Gracilarla tenuistipitata (63), where the external CA facilitates the conversion of HCO," in the medium to COp which then diffuses across the plasma membrane. Conflicting reports have appeared for the red alga Chondrus crispus. Smith and Bidwell (138) reported the presence of external CA, while Giordano and Maberly (61) did not find it to be present. The brown alga Ascophyllum nodosum uses HC0 3 ~ and lacks external CA activity (78). In contrast, external CA activity was found in some brown algae of the Fucales and Laminariales (141). A difference exists with regard to the presence of CA in the green macroalgal genus Ulva. Bjork et al (15) report the presence of CA in U. rlglda as do Giordano and Maberly (61) for U. lactuca, although the activity is low. In contrast, Drechsler and Beer indicate they found no external CA activity for U. lactuca (47). No external carbonic anhydrase activity could be detected on the Udotea thalli by direct measurement. Thus, it is unlikely that a DIC utilization mechanism, if present, would have external CA activity as a component. Reiskind et al (123) measured high levels of internal CA

PAGE 74

61 activity in Ddotea, not associated with the membranes, and thus presumably soluble. Their data suggest that CA is located in the chloroplasts, but the concurrent activity in the cytosol cannot be excluded. Activity of CA in the cytosolic compartment could play a role in maintaining a sufficient C0 2 supply for the PEPCK enzyme that appears to be located in the cytosol (122,123) if HC0 3 be transported across the plasma membrane into the cytosol. The final pH values attained in a pH drift experiment were 8.3 and 8.95, which are in the range of C0 2 ~only users reported by Maberly (94). A low pH compensation point, the pH at which no net carbon exchange occurs, has been observed for sublittoral algae within each division, and this may be related to the growth conditions of these algae (6). Similarly, the low pH compensation point observed for Udotea as compared to the other algae examined by Maberly (94) may be related to the sublittoral growth conditions of Udotea. Maberly (94) indicates that the ability of a species to raise the pH and deplete the total available inorganic carbon varies with the habitat where the species grows. A major contrast exists between the macroalgae that grow in rockpools, which are very effective at removing inorganic carbon and those that are found subtidally in relatively low light, most of which have a more limited ability to use HCO,~. The final C0 2 concentration attained by Udotea conglutlnata of 5 jj.K was 25-fold higher that previously measured C0 2 compensation points of 7 yL C0 2 L" 1 (0.2 pM) for Udotea flabellum (123), which also exhibits C 4 -like photosynthetic features. The pH drift experiment began with 2 mM equivalent to 0.12 mmol Die present in the incubation bottles. Based on a HC0 3 dehydration rate of 0.29 jumol min~ 1 a total of 0.104 mmol C0 2 would have been produced in 6 h in the 60 mL incubation bottles, and assuming all the C0 2 produced was used for photosynthesis, 0.016 mmol DIC would have remained at the end of that time period. However, the DIG measurements at the end of the incubation indicated that 1000 /jH or

PAGE 75

62 0.06 mmol DIC remained in solution. Thus, as the total DIC in the bottle became too low for the alga to photosynthesize, some of the carbon from its CaC0 3 skeleton may have been utilized as a source of carbon to be reduced, perhaps avoiding potentially lethal photoinhibition At the end of the incubation period, 2 of the 3 replicates showed an increase in the alkalinity of the artificial seawater, from 1.36 at the start to 1.93 when a final pH of 8.21 was attained, and 2.58 when the final pH reached 7.89. This phenomenon has also been reported for other macroalgae in pH drift experiments, including Dllsea carnosa, Polyslphonia nigrescens, and Ceramlum rubrum, all red algae (6). These species grow sublittorally, as does Udotea. It has been suggested that an increase in the alkalinity of the seawater was responsible for the excretion of inorganic carbon into the medium by the red algae (6). Such an increase in alkalinity of the water surrounding the thallus could have been caused by the reduction of an internal pool of nitrate (6, 70). Photosynthetic rates for Udotea at pH values of 7.7, 8.2, and 9.0 demonstrated "classic" HC0 3 ~ utilization by the alga (42, 77). Photosynthetic rates in solutions of different pH with the same concentrations of free C0 2 and varying amounts of HC0 3 ~ and C0 3 2 ~ were plotted versus C0 2 concentration. As CC> 3 2 ~ is not used by plants for photosynthesis (93), the increasing concentration of HCO-T relative to C0 2 with pH was important. For a given DIC concentration, the proportion of the total DIC present as CO ? decreases with increasing pH. However, at the higher pH values, the photosynthetic rates were higher than measured at a lower pH for the same DIC concentration, thereby strongly suggesting that the HC0 3 ~ present was contributing to the increased photosynthetic rate observed. Even though there was evidence for some HCO~ use in Udotea, the guestion must still be answered whether the ability of the alga to use

PAGE 76

63 HC0 3 in addition to C0 2 from the surrounding seawater is able to explain the observed C A -like photosynthetic features. The crucial part of this question is whether the alga is able to concentrate internal inorganic carbon above the level of the ambient solution so as to be able to shift the reaction of Rubisco to favor carboxylation and the photosynthetic carbon reduction cycle over oxygenation and photorespiration. To concentrate inorganic carbon internally reguires the expenditure of energy (40) due to the thermodynamic constraints of the system. Photosynthesis in species that can use HC0 3 ~ is substantially more than half-saturated with air-equilibrium DIC concentration, i.e., 10 /jM C0 2 and 2 mM HC0 3 ~, while those restricted to using C0 2 are saturated to a lesser degree at the same DIC concentrations (94). Thus, the degree of saturation for the photosynthetic rate at ambient DIC concentration can be used as a guide to indicate whether a carbon concentrating mechanism is operational. In the red alga Gracilarla conferta, photosynthesis saturates at a DIC concentration close to ambient and exhibits 2 ~insensitive photosynthesis, thus suggesting the operation of some sort of carbon concentrating mechanism, perhaps HC0 3 utilization (73). Although internal DIC concentrations were not measured directly, the lack of saturation of photosynthesis in Udotea at ambient DIC concentration suggested that the alga was using primarily C0 2 for photosynthesis. The data obtained from the effect of 40 mM KI on photosynthesis provided more clues as to whether Udotea can use HC0 3 ~. It is possible that the K + ion interfered with a Na + /HC0 3 symporter in the plasma membrane, the same sort of transporter reported to be operating in the marine diatom Phaeodactylum tricornutum (46). Potassium iodide did not exhibit an inhibitory effect until the concentration was increased to 40 mM, but the same concentration also was used in Ulva lactuca to inhibit photosynthesis at pH 8.2 (47). This is a high concentration, but if it was competing with Na + the concentration of Na + in the bathing medium

PAGE 77

64 was 450 mM, so potentially an inhibitory effect occurred at one-tenth the concentration of the important ion. Another possibility is that the I" ion was in competition with HC0 3 for transport on a membrane translocator. This is an unlikely scenario, as the Cl~ concentration was greater than 450 mM, so it would be more likely to interfere with HC0 3 than I Also, HC0 3 was not able to reduce the inhibition. It cannot be ruled out that the I" concentration inside the siphons may have interfered with various cellular processes. In the presence of 40 mM KI, the pH of the thallus surface was 1.0-1.5 pH units lower than the control, where thalli were immersed in 2 mM DIC in unbuffered artificial seawater, indicating that the KI increased H + extrusion or OH~ uptake by the alga. The pH microelectrode used for these measurements was placed in contact with the thallus, but Udotea conglutlnata has a sheath external to the plant cell wall, and the role and chemical composition of this sheath are unknown (24). Thus, the pH electrode could not be placed immediately next to the cell wall. Nonetheless, the pH data indicated that the thallus was performing either H + extrusion or OH" uptake at the surface. This acidification did not appear to be localized to particular regions, as the lower pH was found all over the whole thallus and on both sides. Polarity with respect to pH, as exhibited by the submerged aguatic angiosperm Potamogeton lucens on opposite sides of the leaves (139), or the green alga Chara in different regions of the cell wall (155) could not be detected. One caveat is that the size of the microelectrode tip (1.5 mm diameter) would preclude the detection of acid/alkaline banding patterns that were smaller than about 1 mm in width. In contrast to these Udotea data, measurements of surface pH of Ulva have indicated that this thallus has a much higher pH of 10, but it is also uniform with no detectable zones of acidification or alkalinization (12). Clearly, Ulva is not extruding H +

PAGE 78

65 The contrast in thallus pH measurements between Udotea and Ulva suggest a difference in the DIC utilization mechanisms. The H + extrusion at the Udotea surface could cause HC0 3 in the boundary layer to be converted to C0 2 which could then either diffuse into the cytosol or be actively transported across the plasma membrane into the cytoplasm. C7Iva, in contrast, is likely to be transporting HCO,~ across the plasma membrane into the cytosol via a HC0 3 ~/OH~ antiport, which would be accompanied by an increase in pH on the outside of the thallus (12). Calcification (100) or a Na + /H + antiport system, as reported for Dunallella salina (82) could be causing the H + release However, when a section of Udotea thallus was incubated in the dark for 15 min, the plant surface was 0.4 pH units lower than the seawater in the incubation medium. Thus, photosynthesis or even the presence of light was not essential for the putative extrusion of H + The pH change at the algal surface caused the artificial seawater pH to decrease by 0.4 units during the time the thallus pH was being measured. In the case where no plant was present and the artificial seawater was incubated in the light, the water pH dropped by 0.1 unit over a 15-min period, probably due to a temperature increase in the water, so most of this acidification of the external water could be attributed to the presence of the macroalga. The inhibition of photosynthesis in Udotea that occurred in the presence of DIDS suggested the presence of an anion exchanger in the plasma membrane. In red blood cells, a protein located in the plasma membrane called the band 3 anion transporter (anion exchanger) facilitates HC0 3 ~ transport, but this is not an active transport system, and thus does not reguire expenditure of ATP energy (53, 135). In red blood cells, this protein catalyzes the transmembrane exchange of one HC0 3 ~ for one CI". The inhibitor DIDS, used in this study of Udotea, competes guite specifically with cl" for binding at a lysine residue in

PAGE 79

66 the vicinity of the outward-facing transport site and inhibits inwardfacing sites by shifting their conformation to that of the outwardfacing site (53). This band 3 anion transporter is also located in the plasma membrane of Ulva, as DIDS was able to reduce the photosynthetic rate when HC0 3 but not C0 2 was available as the external DIC species to be taken up by the alga (47). In the presence of DIDS, the surface pH of the alga was 0.2 to 0.3 units more acidic as compared to the control, although H translocation is not reported as a function of the anion exchanger. Based on data from thallus incubation with vanadate, ATP may be necessary for utilization of HC0 3 ~, as after 2 h incubation in 5 mM Na 3 V 4' Photosynthesis was inhibited 34%. For intact cells of Dunaliella parva, where a plasma membrane ATPase plays a role in photosynthesis, 77% of the ATPase activity was inhibited after 3 h incubation in vanadate (60). For intact Scenedesmus and Chlamydomonas cells, vanadate inhibited plasma membrane ATPase activity after 90 min incubation (114). In the presence of vanadate, Udotea thallus surface pH remained unchanged over time, and the pH of the water did not decrease, suggesting that the putative H + extrusion was eliminated. These data may lend further support for the presence of a plasma membrane H + translocating ATPase. If the ATP synthesis is halted, no proton movement occurs, as the two processes are intimately tied together, according to the chemiosmotic hypothesis. Thus, if a HC0 3 ~ translocator is operating in Udotea, it appears to reguire ATP for the transport and so be able to accumulate DIC inside the cell at a concentration higher than that found in the surrounding medium. However, after the 2 h incubation with vanadate, photosynthesis was decreased only 34%, thus suggesting that other DIC utilization mechanisms were operating to maintain the photosynthetic rate. A comparison was made between the measured rate of photosynthesis and that predicted if photosynthesis was limited by the rate of C0 2

PAGE 80

67 production from the spontaneous dehydration of HC0 3 ~. This analysis is complicated by the process of calcification that occurs in Udotea conglutinata. In Chara, it has been demonstrated that photosynthesis and calcification occur in about a 1:1 ratio (99). Using 2 evolution as a measure of carbon metabolism in photosynthesis assumes that for every mole of inorganic carbon fixed, one mole of 2 is evolved. But when considering the effect of calcification in this picture, it must be considered that when the alga is photosynthesizing, each photosynthesis "event" reguires the uptake of 1.25 moles of inorganic carbon: one is fixed by Rubisco in the chloroplast and after four fixations, precipitation occurs at the surface with Ca 2+ to form carbonate (23). Inorganic carbon use in Udotea in light of these two processes must then be considered. At 2 mM DIC and pH 8.2, the ambient conditions found in natural seawater, 0.58 ;umol of C0 2 was produced per hour in the closed system of the 2 electrode chamber based on the kinetics of HC0 3 ~ dehydration. In 21% 2 the photosynthetic rate was 0.44 jumol h~ 1 indicating that C0 2 was supplied at a rate that exceeded the uptake rate. If calcification is considered in addition to the measured 2 evolution, then the carbon really used by the alga for photosynthesis was 0.55 /jmol h" 1 The C0 2 supply rate of 0.58 jumol h~ 1 still met the inorganic carbon reguirements of the algae. At 2 mM DIC and 2% 2 the photosynthetic rate was 0.78 jLimol h~ 1 Under these conditions, C0 2 could not be produced fast enough to meet the photosynthetic reguirement, so HC0 3 ~ use may have made up the deficit. In 1 mM DIC with a HC0 3 dehydration rate of 0.29 ^mol h" 1 under both 21% and 2% 2 HC0 3 would have to be used to meet the reguirement for photosynthetic carbon and then additional HC0 3 used for calcification. When the alga was placed in 5 mM DIC, more than twice the concentration the plant is exposed to in nature, C0 2 was supplied from HC0 3 dehydration at 1.46 jumol h _1 a rate higher than the 2 evolution in both 21% and 2% 2 thus indicating that the CQ 2 supply was sufficient for both carbon fixation

PAGE 81

68 and calcification. Thus, it appears that Udotea does have some capacity for HCO,~ utilization above and beyond the use of free CO.,. Although 2 inhibited the photosynthesis of Udotea, it was unlike the phenomenon observed in C, plants. The photosynthetic characteristics of Udotea f label lum clearly indicate that its metabolism is C^-like in nature (123), and some earlier work on U. conglutlnata indicated the same metabolism as the other species (Reiskind & Bowes, unpublished) The apparent 2 sensitivity of photosynthesis may be attributed to other 2 effects, such as the Mehler reaction, mitochondrial respiration, or the glycolate pathway, similar to that found in the red alga Chondrus crispus where 2 inhibition is relatively insensitive to C0 2 concentration and not completely reversed by high C0 2 (30) Because Udotea conglutlnata is a calcareous alga, the skeleton of carbonates plays a role in photosynthesis in addition to providing structural integrity for the intertwined siphons of the thallus. In species that calcify, the process does not occur until the cells are photosynthetically competent (24). In the presence of CaCl 2 at 2 mM DIC and 21% 2 twice as much DIC was removed from the medium as 2 was evolved. This proportion suggested that 2 moles of inorganic carbon were required to complete the whole photosynthetic process in Udotea of 2 evolution and calcification. At 5 mM DIC, nearly 10 times more DIC was removed from the medium as 2 was evolved, but the quantity of 2 evolved was approximately equal to that in 2 mM DIC. A portion of this inorganic carbon could be used in calcification, but the reason for this difference is not known at this time. When CaCl 2 was withheld from the photosynthetic medium, photosynthesis was measurable as 2 evolution in the 2 electrode, but the amount of inorganic carbon in the medium actually increased during the course of the 15 min measuring period. Thus, it may be that the plant was excreting inorganic carbon at its

PAGE 82

69 surface which normally would complex with Ca 2+ to form CaCO, in the skeleton. Chara also does not calcify under low Ca 2+ conditions (99). Calcification in Udotea appears to occur by a mechanism similar to that in Halimeda, a closely related siphonaceous species from the same family (23). Calcium carbonate precipitates as aragonite crystals in the intercellular spaces which are separated from the external seawater. In Halimeda, swollen ends of the siphons, called utricles, cause this isolation and the sheath found outside the cell wall in Udotea conglutinata may serve a similar function. The model for Halimeda states that about 70% of the cell wall area faces these intercellular spaces. When C0 2 uptake occurs from the carbon in the intercellular spaces, the pH increases as does the concentration of CO, 2in these secluded spaces and Ca from the surrounding medium moves through the cell walls to these areas, thereby facilitating CaCO-, precipitation (23). The photosynthetic removal of carbon is greater than the resupply rate from the external seawater and cellular respiratory processes. The surface acidification in the presence of CaCl 2 in the seawater is a byproduct of the precipitation of CaC0 3 (100). Studies in Chara also have provided important information about the calcification process. Protons generated from the hydroxylation and precipitation of C0 2 allow the plant to manufacture twice the amount of C0 2 used in calcification (99). The model for Chara suggests that the inorganic carbon and Ca 2+ for the process come from inside a heavily encrusted plant, and a Ca /ATPase appears to be involved in part of the transport (99). The H + must be removed from the calcification site; perhaps H + are transported by cytoplasmic streaming and through a large, acidic vacuole to another part of the plant cell. In fact, H + generation may be the principal benefit of calcification (99). This precipitation occurs at an appreciable rate in nature, as 50% of the dry weight of the Udotea thalli was composed of carbonate salts, and a similar proportion occurs in Characeans (100).

PAGE 83

70 Calcification in Udotea could occur in the intercellular spaces, which are really spaces between the siphons. As CaCO, precipitation occurred, the H + generated were translocated to the outer surface of the thallus, and this H + extrusion was recorded in this investigation. The calcification process was actually quite similar to that for Chara, with H + movement occurring across the same cell, or siphon in this case, except that only the acidification could be measured because the removal of H + occurred inside the thallus. However, in Udotea, some H + extrusion continued in the absence of photosynthesis. The current hypothesis regarding inorganic carbon utilization in Udotea is that the alga preferentially uses C0 2 when it is available, but limited HCO,~ use also occurs. The HCO, is probably transported by a carrier protein in the plasma membrane in a facilitated manner, such that no energy expenditure is required, and thus internal accumulation of DIC does not occur above the external concentration. However, it also appears as if some ATPase is involved, but no external CA. ATPase may be involved in calcification or conversion of HC0 3 ~ to C0 2 by acidification and thus H + extrusion. Thus, the model for photosynthesis in Udotea includes DIC uptake in the form of C0 2 which is produced in the boundary layer by H + extrusion associated with the calcification process. This uptake could occur via diffusion or active transport of the CO., across the plasmamembrane. Some HC0 3 ~ uptake across the plasmamembrane also occurs probably on an anion exchanger that operates via facilitated transport. As inhibition of these mechanisms inhibited photosynthesis by no more than 34%, the high phosphoenolpyruvate carboxykinase activity, reportedly located in the cytosol, is the key component of the photosynthetic mechanism in Udotea to concentrate inorganic carbon for Rubisco and thus, cause the macroalga to exhibit its C^-like features.

PAGE 84

CHAPTER 3 ROLE OF PHOSPHOENOLPYRUVATE CARBOXYKINASE IN THE PHOTOSYNTHESIS OF THE MARINE MACROALGA UDOTEA Introduction Udotea is a green, marine, macroalga which exhibits C,-like photosynthetic characteristics, despite lacking the Kranz anatomy associated with terrestrial C, species. The thallus of this alga is composed of intertwined coenocytic siphons. Although a "primitive" plant, this alga appears to utilize a more "advanced" type of photosynthetic metabolism, initiated by the enzyme PEPCK, as when PEPCK is inhibited by MPA, the photosynthetic metabolism of this alga appears C 3 -like (122). The focus of this investigation was to examine the kinetics of PEPCK in order to ascertain how this enzyme could be functioning as a component of the C^-like metabolism. In some higher plants that utilize C^ photosynthesis, PEPCK functions as a decarboxylase in the cytosol of bundle sheath cells (4). In the brown algae, PEPCK is hypothesized to function in an anapleurotic role to supply the cells with carbon skeletons for the synthesis of other compounds (159). PEPCK is also found in a variety of other organisms ranging from bacteria and yeast to diatoms and vertebrates. In the plants and diatoms, PEPCK uses ADP/ATP, while the enzyme in Euglena and vertebrates utilizes GDP/GTP for its activity (117, 152). In mammals, PEPCK is a critical enzyme in gluconeogenesis, involved in removing C0 2 from OAA to produce PEP for the eventual production of glucose. Materials and Methods Materials The marine macroalga Udotea f label lum (Ellis and Solander) Lamouroux (division Chlorophyta) was collected during the summer on the north side of Marathon Key, Florida, in Florida Bay in water ranging 71

PAGE 85

72 from 0.5-2.5 m in depth. After collection, the plants were kept outdoors for up to two days in a fresh seawater-fed and aerated tank until frozen in liquid nitrogen (LN 2 ) Enzyme Extraction For experiments on crude extract, 1-2 g of LNp-frozen plant material was ground in a mortar and pestle with sand at 4C in 3-6 mL of 50 mM Hepes-NaOH, 2 mM MnCl 2 5 mM DTT, 1% (w/v) PVP-40, 1 mM PMSF, and 10 pM leupeptin at pH 7.0. The homogenate was centrifuged at 13,000 g for 2 min in a microfuge at 4C. The supernatant was then desalted through either a 5 mL Sephadex G-25 or BioRad P-6 column with the same medium used for grinding, but without PVP-40. The column eluant collected just after the void volume was then used for subsequent enzyme assays. For isolation of Udotea PEPCK, approximately 45 g of frozen plant material was powdered in LN 2 and then ground with a mortar and pestle and sand at 4C in 150 mL of a medium containing 50 mM MES-NaOH, 5 mM MnCl 2 5 mM MgCl 2 5 mM DTT, 2 mM Na 2 EDTA, 1 mM PMSF, and 1% (w/v) PVP-40 at pH 6.5. Following centrifugation at 4C for 15 min at 12,000 g, the pellet was discarded, and the supernatant was used for protein isolation. Solid (NH 4 ) 2 SC> 4 was added slowly, with constant stirring, to the protein solution kept on ice. The protein that precipitated between 40 and 50% (w/v) (NH 4 ) 2 S0 4 was pelleted at 3500 g and resuspended in 6 mL of the elution buffer, which consisted of 50 mM MES-NaOH, 5 mM MnCl 2 5 mM MgCl 2 5 mM DTT, and 1 mM PMSF at pH 6.5. The solution was then brought to 20% (v/v) glycerol. Enzyme Purification The protein solution was loaded onto a Sephacryl S-300 column (78 x 2.5 cm, 380 mL bed volume) and eluted at 4C with the buffer described above. Fractions were analyzed for protein by measuring the absorbance at 280 nm in a spectrophotometer. Fractions were also analyzed for PEPCK, malate dehydrogenase (MDH) and Rubisco activities. Those

PAGE 86

73 fractions containing high PEPCK activity and low MDH activity were loaded onto a Sepharose CL-6B column (25 x 1.5 cm, 40 mL bed volume) for ion exchange chromatography and eluted with a linear 0-0.3 M KC1 gradient in the same buffer used for gel filtration. Fractions were analyzed for PEPCK, MDH, and Rubisco activities, and protein. The fractions containing highest PEPCK activity were pooled (total volume 35 mL) the protein concentration was determined, 2 mg mL bovine serum albumin (BSA) was added, and the solution was frozen in LN 2 for further enzyme studies. Enzyme Assays Enzymes were assayed at 25C (except where noted) The carboxylation activity of PEPCK was routinely measured spectrophotometrically at 340 nm by the MDH/NADH-coupled reaction (151). The standard assay mixture consisted of 50 mM PIPES-NaOH, 5 mM MnCl 2 30 mM NaHC0 3 5 mM DTT, 20 to 30 fiL enzyme extract, 2 units MDH, 0.2 mM NADH, 3 mM ADP, and 10 mM PEP at pH 6.8 in a total volume of 0.5 mL. The reaction was initiated by adding ADP and followed for a period of 3 minutes during which the reaction proceeded at a linear rate. An absorption coefficient of 6.22 mM -1 cm" 1 was used for NADH. The PEPCK carboxylation activity was also measured radiochemically in a standard assay of 50 mM PIPES-NaOH, 5 mM MnCl 2 30 mM NaH 14 C0 3 (2 pCi prnol" 1 ) 5 mM DTT, 20 to 30 /jL enzyme extract, 2 units MDH, 0.2 mM NADH, 2 mM ADP, and 7 mM PEP at pH 6.8 in a total volume of 0.5 mL. The reaction was initiated with PEP and terminated after 6 min with 0.1 mL 6 N HCl saturated with 0.13 M dinitrophenylhydrazine. After drying at room temperature to allow unreacted NaH 14 C0 3 to escape, the sample was rehydrated with 0.4 mL deionized water, one drop of 6 N NaOH, and 4 mL liquid scintillation cocktail (RPI 3a70). The capped vials were then vortexed. The acid-stable products were counted in a liquid scintillation counter.

PAGE 87

74 The decarboxylation activity of PEPCK was measured spectrophotometrically at 340 nm by the MDH/NADH-coupled reaction modified from Burnell (33). The assay mixture consisted of 50 mM Bicine-NaOH, pH 7.8, 5 mM MnCl 2 20 to 30 pL PEPCK enzyme extract, 0.25 mM NADH, 1 unit lactate dehydrogenase, 2 units pyruvate kinase, 0.5 mM ATP, and 0.5 mM OAA, at pH 7.8, also in a total volume of 0.5 mL. The reaction was initiated with OAA and followed for 3 min, during which time the reaction proceeded at a linear rate. The activity of MDH was assayed spectrophotometrically at 340 nm by measuring NADH oxidation in a method modified from Yueh et al (161). The assay mixture was composed of 50 mM Bicine-NaOH, 1 mM Na 2 EDTA, 0.2 mM NADH, 10 /jL enzyme extract, and 0.5 mM OAA at pH 8.0. The reaction was initiated with OAA. The activity of Rubisco was measured radiochemically in an assay mixture consisting of 50 mM Bicine-NaOH, 10 mM MgCl 2 50 y.~L enzyme extract, 5 mM DTT, 5 mM isoascorbate, 10 mM NaH 14 C0 3 (0.5 /iCi /jmol -1 ) and 1 mM RuBP at pH 8.0. The enzyme was activated for 5 min at 30C in the assay mixture without RuBP present (128). The reaction was initiated with RuBP, performed at 30C, and terminated after 30 s with 6 N HC1. After drying in a 70C oven and rehydrating the samples, the radioactivity of the acid-stable products were measured as described above Protein Assay During elution of the columns, protein concentration was determined by measuring the absorbance of the protein solution at 280 nm, but in all other instances, it was determined by the method of Bradford using bovine T-globulin as standard (29). Enzyme Storage Conditions Following purification, aliguots of PEPCK were stored for a period of 5 weeks as enzyme solution in elution buffer, with and without 2 mg mL -1 BSA, or as the precipitate of 65% (NH 4 ) 2 SO^ in the refrigerator

PAGE 88

75 (4C), freezer (-20C), or in LN 2 (-196C). The carboxylation activity of the aliquots was measured after 2 weeks and again after 5 weeks of storage. Enzyme Kinetic Analyses The inorganic carbon species utilized for carboxylation by Udotea. PEPCK was determined by the method of Cooper et al (44) at 12C so as to slow equilibration among the inorganic carbon species. The DIC was added at a concentration of 8 mM as either HC0 3 ~ or C0 2 to initiate the reaction in the presence or absence of CA. To provide predominantly HCOj" to the enzyme, a solution of 0.5 M NaHC0 3 was added directly from a Hamilton syringe into the assay mixture in the cuvette. However, to provide predominantly C0 2 NaHCOj solution was drawn up into a Hamilton syringe followed by an equal volume of 0.5 N HC1. After dissolved C0 2 was produced in the syringe, the solution was dispensed into the cuvette where the reaction was to occur. The reactions were performed at pH 8.0. All solutions, except NaHC0 3 were prepared C0 2 -free by bubbling with N 2 This bubbling was continued after the mixture components, except for the DIC, were combined in the cuvette, so that the assays were performed under C0 2 ~free conditions. The DIC was added last to initiate the reaction. The reaction was followed for only one minute in the spectrophotometer so that the measured rate would be dependent upon the inorganic carbon species initially added to the reaction mixture. The affinity of PEPCK for the carboxylation substrates, DIC, ADP, and PEP and the decarboxylation substrates, OAA and ATP was examined. The effect of Mn 2+ was investigated for both reactions. The concentration of the substrate in question was varied while the other reaction mixture components were held constant at saturating concentrations. The constants were calculated from Eadie-Hof stee plots. The computer program Enzfitter was also used to calculate MichaelisMenten constants.

PAGE 89

76 The carboxylation reaction was performed in the presence of ADP, GDP, or IDP, and the decarboxylation reaction was run with ATP, GTP, or ITP to determine how these components affected the reaction. The effects of 0.5 mM OAA and 0.5 mM ATP on the carboxylation reaction and 1 and 3 mM DIC, 0.02 mM ADP, and 4 mM PEP on the decarboxylation reaction were examined. For both carboxylation and decarboxylation, the effect of 100 /jM MPA on Udotea PEPCK was investigated. The effects of the intracellular metabolites malate, aspartate, F6P, F-1,6-BP, 3-PGA, and dihydroxyacetone phosphate (DHAP) were examined for both reactions. These potential effectors were added at 4 mM over a range of subsaturating concentrations for one reaction component at a time. Other substrates were present in saturating concentrations The carboxylation reaction was run at subsaturating DIC concentrations and the decarboxylation reaction at subsaturating OAA concentrations in 0, 21, and 100% gas phase 2 (0, 0.46, and 2.18 mM 2 in solution) to determine whether 2 was an effector of either the carboxylation or decarboxylation reactions. Results In initial experiments, assays of PEPCK activity in both the carboxylating (pH 6.8) and decarboxylating (pH 7.8) directions were performed with crude extracts of Udotea thalli subject only to gel filtration through a 5 mL column of Sephadex G-25. The V mgx values from the preparations ranged from 0.69 to 1.32 ^mol NADH mg -1 protein min -1 and 0.51 to 0.98 jumol NADH mg -1 Chi min -1 for the carboxylation reaction, but were approximately fourto five-fold lower for the decarboxylation reaction, ranging from 0.14 to 0.34 /^mol NADH mg -1 protein min and 0.10 to 0.25 /jmol NADH mg -1 Chi min -1 The K m values for the various substrates are listed in Table 3-1. In the carboxylating direction, the enzyme exhibited an almost 100-fold concentration difference in K ffl values, with the lowest K being for ADP and the highest for dissolved

PAGE 90

77 Table 3-1. K m values for PEPCK activity in crude extracts of Udotea thalli. The values were derived from Eadie-Hof stee plots. Substrate K m mM Carboxylating Direction ADP 0.075 PEP 0.920 DIG 7.326 Decarboxylating Direction ATP 0.023 OAA 0.028

PAGE 91

78 inorganic carbon. In contrast, the affinities of the substrates ATP and OAA for decarboxylation were similar and comparable to the low K (ADP) for carboxylation. In an experiment where the same concentration (5 mM) of GDP or IDP was substituted for ADP, carboxylation proceeded at less than 20% of the ADP-catalyzed rate (Table 3-2). Similarly, for the decarboxylation reaction, the rate with 0.4 mM GTP or ITP was considerably reduced when compared to the rate with the same concentration of ATP. The nucleotide preference of the decarboxylation reaction was less specific than that of the carboxylation reaction, as the decarboxylation rate proceeded more rapidly when other nucleotides were substituted for ATP. An examination of the metal ion requirement for PEPCK in crude extracts indicated that Mn was essential for maximum catalytic activity in the carboxylation direction (Fig. 3-1). When Mg 2+ was supplied at the same concentration as Mn 2+ catalysis was only 10 to 25% of the rate with Mn 2+ ; thus, Mg 2+ alone could not substitute for Mn 2+ If both species were present at the same concentration, Mg 2+ inhibited the rate obtained in the presence of only Mn 2+ by 5% and 44% at 2 and 5 mM ion concentration, respectively. Udotea PEPCK was purified in a three-step procedure involving (NH^JjSO^ precipitation, which removed a portion of the extraneous cellular proteins, followed by separation of the proteins of interest according to molecular weight using gel filtration, and finally an ionexchange column. Protein was measured by reading the absorbance at a wavelength of 280 nm, and the presence of PEPCK was detected by its carboxylation activity. Two other enzymes, MDH and Rubisco, were also detected by assay of their activity in various fractions. The gel filtration step separated PEPCK from most of the MDH, but some MDH activity remained (Fig. 3-2). This step also eliminated the remainder of the Rubisco activity associated with the PEPCK. For the next step in the purification procedure, only the PEPCK fractions preceding the first

PAGE 92

79 Table 3-2. Effect of various nucleotides on crude PEPCK activity. The carboxylation rate was 0.642 0.018 /jmol NADH mg _1 protein min "^ with ADP, and the decarboxylation rate was 0.299 0.021 /iffiol NADH trig -1 protein min 1 with ATP. Relative Reaction Rate Substrate Carboxylation Decarboxylation % ADP 100 GDP 17 IDP 10 ATP 100 GTP 38 1TP 55

PAGE 93

80 c O *-> (0 l +-* c p -p n QJ 1S1 03 u a (1) a -rl £ -P • o ED Q -P o H cd o U -P ,— ^ X 0) (1) + o s 3 O u e o h M-l — >i -P <\J -H c > E H +1 S4 O 0) m £ -p c •w 0) H 0 m a r-t >, -P X u ~Ik, —

PAGE 94

81 082V o o O o O o o o L0 o LO o LO o n CM OJ 11o o ewAzue t _~|iu k _uiuj ptur/ i-l a o (1) l-l o £ O £ P P & C o o o id < T3 — Q) 3 U CD ra rd £ Si 3 en — c M o c u o Hi C H -H 3 CU 0) -P LU CO tx, •H CD P o • u c & £ 3 X! P m o o o O £ O P ro CO £ o o >i U U T3 ao o) m p c -a 3 O Q|H H 0) CD p w M ro M-l M-l CD P
PAGE 95

82 major MDH peak (i.e., elution volume 130 through 210 mL) were selected. These fractions with high PEPCK activity were pooled and loaded onto the ion exchange column. After rinsing with buffer lacking KC1, elution was accomplished with a 0-0.3 M linear KC1 gradient in the elution buffer. Ion exchange reduced the proportion of MDH in the PEPCK fraction such that the pool of the most active PEPCK had an average activity ratio of 7 units PEPCK to one unit of MDH (Fig. 3-3). This step also separated the remaining Rubisco activity from the PEPCK. After gel filtration and ion exchange chromatography, PEPCK was purified 12.6-fold, resulting in a specific activity of 18.52 pmol NADH mg -1 protein min -1 (Table 3-3). To test its stability and determine optimum storage conditions, the isolated enzyme was stored under a variety of conditions for up to five weeks. Three temperatures were utilized (4, -20 and -196C) and the enzyme was maintained in the ion exchange elution buffer or with 2 mg mlf 1 BSA, or as precipitate in 65% (w/v) (NH 4 ) 2 S0 4 (Table 3-4). After 2 weeks, the enzyme stored as a solution in buffer or as a precipitate with (NH 4 ) 2 S0 4 at 4 and -20 C lost nearly all activity, whereas that kept in LN 2 at -196C still retained about one-third of the initial activity. The enzyme stored with BSA retained between 70-90% of its initial activity at all the temperatures. After 5 weeks, the enzyme stored with BSA at -20C had lost half of its original activity, while the samples stored at 4 and -196C retained 66% of the initial activity. Based on these experiments, the purified enzyme was routinely stored at -196C after adding 2 mg mL -1 BSA to the solution, and this solution was utilized to characterize the purified enzyme. When measured as a function of pH, the carboxylating and decarboxylating activities of isolated PEPCK exhibited peaks of activity ranging from 6.0 to 7.5 and 7.5 to 8.0, respectively (Fig. 3-4). At pH 7.6, the rates of the two activities were eguivalent, but at its optimum pH of 6.8, the carboxylation rate was 136% of the decarboxylation rate at its optimum of pH 7.8.

PAGE 96

83 !3>1 Iaj pue 082V £ C o 3 LLi o o O o o o If) CM 01 cO en o y— T— o o o o eiuAzue k _"~[UJ ,_U]LU |Oiu7? a e 3 P rCfl d Q) 0) Oi-H u -a 0) cd cd x; J3 vi -P en g H -p O u M-l en C2TJ -r) 0) T3 ro -P H • 3 OOrl cu (I) -P CO a — 1 o < r) """ w cd o • s cu c M 6 +J Cu 3 cd 01 "O c o CD •rl V! CU 3 -P M3 0! 1 ca u J CU ftO £ CU 0) en X! CD cd -p u 9 m rd — o x:< a ~— 0) CU .h CO a •H M-l CU -h £ -p n -p cd ft V] e -p a c u CU •H M-l u -P G 3 m o r-H 0) o H H 3 c o 1 O m S VI a, CD CU Vl si 3 p • & — -H en fe

PAGE 97

(8 O •H -rH cm 0) > o u CD 84 c Q) -p M Cm CD 6 .-H > c o -.H -p •, en 4-1 -p £ r •H -H 01 • CD -A -P tH ft-P •H W O C rt 3 CM in 2 * CN 01 LD m CN a CN <0 U3 PCNJ in CO in 00 -a 01 T3 rH >1 m u o o J^w ta o o H CQ 01 n) o nJ ix> T) in ~*h 3 £ ro A I 3 i IE rH in ft I & j U O S CD CD CD W CD CJ U 3— ft U W OT

PAGE 98

85 Table 3-4. activity. Effect of storage conditions on purified PEPCK carboxylation he initial activity was 15.17 umol NADH mg pro min ", The Storage Conditions Temperature Time Addition c Weeks 4 2 Buffer + BSA +(NH 4 ) 2 S0 4 4 5 Buffer + BSA + (NH 4 ) 2 S0 4 -20 2 Buffer + BSA + (NH 4 ) 2 S0 4 -20 5 Buffer + BSA + (NH 4 ) 2 S0 4 -196 2 Buffer + BSA + (NH 4 ) 2 S0 4 -196 5 Buffer + BSA + (NH 4 ) 2 SQ 4 Activity Remaining pimol NADH mg -1 pro min 1 % 0.42 3 13.83 91 0.67 4 0.12 1 10.00 66 0.32 2 1.69 11 10.42 69 0.77 5 0.60 4 7.68 51 1.43 9 4.88 32 13.42 88 4.21 28 1.84 12 9.86 65 3.37 22

PAGE 99

86 I Q. _uiuu ojd t _5oj HQVN puury -a Q) •H M-l H M a M-l m a) + H > -rH -P c o --I H >i X o (d D o T3 C o A -P id H S n u a o -p a o M-l -P o 0) 14-] m H u o • Ln m +j a) 3 O DiCM •r4 W fa Pn

PAGE 100

87 The carboxylating and decarboxylating reactions of PEPCK exhibited a similar optimum temperature of 35C when measured at their respective pH optima (Fig. 3-5). The carboxylation reaction for the purified enzyme was measured as a function of DIC concentration at the optimum pH for carboxylation, 6.8, and at the optimum pH for decarboxylation, 7.8 (Fig. 3-6). The curve at pH 7.8 is flatter than that at 6.8; however, at both pH values, the reaction was saturated at 30 mM DIC with a K 05 (DIC) of 7.8 mM at pH 6.8 and 15.5 mM at pH 7.8. In solution, DIC exists as free C0 2 H 2 C0 3 HC0 3 ~, and C0 3 2_ with the relative proportions dependent upon the pH and temperature of the solution. The relative species proportions were calculated from the Henderson-Hasselbach equation. The pK g/] of 6.36 for carbonic acid (H 2 C0 3 ) in freshwater at 25C was used for the calculations (146). At pH 6.8, 0.273 of total DIC is C0 2 and 0.727 is HC0 3 ", and at pH 7.8, 0.035 of total DIC is C0 2 and 0.965 is HC0 3 ". At these pH values, the proportion of C0 3 2_ was negligible. Using these proportions, it was possible to calculate the Kg 5 values for C0 2 and HC0 3 at the two pH values used in the assay. At pH 6.8, using a Michaelis-Menten plot (Enzfitter program), the K Q5 (C0 2 ) was calculated to be 2.1 mM and the K 5 (HC0 3 ") was 5.6 mM, while at pH 7.8 the K Q 5 (C0 2 ) was 0.5 mM and the K 05 (HCO 3 ~) was 15.0 mM. Thus, the Kq 5 (C0 2 ) at pH 7.8 was four-fold lower than at pH 6.8, whereas the K Q 5 (HC0 3 ") was 2.5-fold lower at pH 6.8. Further investigation of the carboxylation reaction showed that it proceeded most rapidly when free C0 2 rather than HC0 3 ~ was provided to the enzyme (Table 3-5). This assay was run at 12C to reduce the rate of spontaneous equilibration between C0 2 and HC0 3 ~, and the reaction rates were lower as a result of the decreased temperature. Adding CA to speed the equilibration between C0 2 and HC0 3 ~ decreased the carboxylation rate by 36% when C0 2 was the inorganic carbon source. When HC0 3 ~ was the predominant form of C. supplied to the enzyme, the reaction rate was just 28% of that in the presence of C0 2 Adding CA in

PAGE 101

88 2 3 J (0 $ a E L _uiai cud t _5uu HQVN punT •a G >, w ~H > -^ p o (0 c H +J id t-\ !x X ,2 ^
— &o s -P >i +J tW -H > -H +J P o 1 m X d) £ S-l Ul 3 f3 en u h fc '0

PAGE 102

89 O Q n o o o o o N iO in 1 X ja b id 0) C 4J -P tO >< 4J •H > --I P O id c •H +J id <-{ >, x 5 • H —
i Q X M-l a u Id 0) O .rH 4-1 fa

PAGE 103

90 Table 3-5. Form of dissolved inorganic carbon utilized by Udotea PEPCK for carboxylation. The reaction was performed at 12C to slow the equilibration between CO ? and HCO-^~ in solution. Treatment Rate of PEPCK Reaction With CO., With HCO, —1 -1 pmol NADH mg pro min Without CA 9.47 0.51 2.66 0.81 With CA 6.06 0.32 7.44 0.56

PAGE 104

91 the presence of HCO^ increased the rate to 79% of the C0 2 rate. These data indicate that Udotea PEPCK uses C0 2 rather than HC0 3 as the inorganic carbon substrate for carboxylation. The K values for the substrates of carboxylation and decarboxylation are summarized in Table 3-6 for the isolated PEPCK by way of comparison with the crude extract data (Table 3-1). For the carboxylation reaction, the K n (DIC) of 7.7 mM for the isolated enzyme at pH 6.8 was similar to the value obtained with the crude enzyme extract. For the other substrates of the carboxylation reaction, the K m values found with the isolated enzyme were comparable to those observed with the crude extract. However, in the decarboxylation reaction, the K m (ATP) of 2 pM for the isolated enzyme was ten-fold lower than that of the crude extract. A comparison of K values for both reactions indicated that the affinity of Udotea PEPCK for the substrates of the decarboxylation reaction was higher than for the substrates of the carboxylation reaction. Since CO, appeared to be the inorganic carbon substrate for PEPCK, the reactions were examined at several 2 levels to investigate whether Op was a competitive inhibitor with respect to C0 2 as occurs with Rubisco (26, 90). The carboxylation reaction was examined with a range of DIC concentrations from subsaturating to saturating with 0, 21 and 100% 2 in the gas phase (Table 3-7). No inhibitory effect of 2 was observed at any of the DIC or 2 concentrations employed. Similarly, the decarboxylation reaction was insensitive to 2 at saturating and subsaturating OAA concentrations (Table 3-7). 3-Mercaptopicolinic acid has been reported to be a specific inhibitor of PEPCK isolated from mammalian livers and kidneys (120) and from the liver fluke (88). In regard to Udotea, MPA inhibited both reactions competitively: with respect to PEP for carboxylation (Fig. 37) and with respect to OAA for decarboxylation (Fig. 3-8). In the presence of 100 pM MPA, the apparent K (PEP) increased seven-fold, from

PAGE 105

92 Table 3-6. K values for substrates of isolated PEPCK at optimal pH for the reactions'" determined from Eadie-Hof stee plots. The K m values for C0 2 and HCO," at each pH were calculated based on the HendersonHasselbach equation using the pK g1 of 6.36 for carbonic acid in freshwater at 25C (146). Reaction Substrate PH K„, mM Carboxylation Decarboxylation DIC co 2 HC0 3 DIC co 2 HCD 3 PEP ADP Mn 2+ OAA ATP Mn 2+ 6.8 6.8 7.8 7.8 7.8 6.8 6.8 6.8 7.8 7.8 7.8 7.70 2.10 5.60 16.62 0.58 16.04 1.66 0.03 0.88 0.035 0.002 1.67

PAGE 106

93 Table 3-7. Effect of oxygen in the gas phase on the carboxylation and decarboxylation activities of isolated PEPCK at various DIC and OAA concentrations. Reaction and Substrate Rate of PEPCK Reaction Concentration 0% O-, 21% CX, 100% O, mM Carboxylating (DIC) 2 1.61 0.07 5 5.81 0.41 8 8.66 1.12 20 13.18 1.12 30 17.14 0.76 Decarboxylating (OAA) 0.05 10.50 0.42 0.5 15.37 1.62 pmol NADH mg 1 pro min 2.53 0.55 4.84 0.56 8.55 1.10 13.85 0.41 16.02 0.59 3.44 0.14 5.01 + 0.95 8.42 0.57 14.06 1.45 18.36 0.97 11.07 1.44 15.77 1.57

PAGE 107

94 Q. LU Cl ^(j.UIUJ cud t _6uj HQVN \ouari) Cm P? CM o 4J p o 0) ft CD CD u si -p H 2 §3 a. o o -P •H > 4J cti C O -H 4J fd H | O XI n 6 cm H • Cm -P C m d) to a) u ft c o p XI c •H a) > 4-> — P Q) 4J ft a £ 0) o w O ro 0) U ft cm o c n C7~ fa —

PAGE 108

95 < < o t _( t _U!UJ OJd t _6uu HQVN \ovjrf) 5 -p o % w Qi U *i +j ff 1 s a. o o > •H +U C -H -P (TS X C) A n it) C) a) • Ti -P c w cu O ra ft a) W rH a, Si C H (1) P 01 -H (I) -P u (!) ih ti S rt o 0, D 2 • c 03 (>• a) rH 3 • & % fe o

PAGE 109

96 1.6 to 9.0 mM, while the apparent K m (OAA) increased 3.5-fold, from 41 to 114 iM. A number of cellular metabolites were also examined as potential effectors of the carboxylation and/or decarboxylation reactions of Udotea PEPCK (Table 3-8). The C 4 acids malate and aspartate had only a minor inhibitory effect on either reaction, of no more than 10%, but malate decreased the K m (DIC) from 5.91 mM to 2.97 mM (Fig. 3-9). Malate also lowered the V max by 60%. The PCR cycle intermediates DHAP and 3PGA inhibited PEPCK carboxylation at subsaturating DIC but not at subsaturating PEP. The K (DIC) was decreased by DHAP from 5.91 mM to 3.15 mM, and the V max was decreased by 45% (Fig. 3-10). With respect to DIC, carboxylation activity was inhibited non-competitively by 4 mM 3PGA, and the V mgx of the reaction decreased 45% in its presence (Fig. 311). Two 6-carbon sugar phosphates of the PCR cycle, F6P and F-1,6-BP, also affected the PEPCK reactions. Fructose-6-P inhibited decarboxylation by 30% at subsaturating OAA concentrations while it slightly stimulated carboxylation, by 14%, at subsaturating levels of PEP. At a concentration of 4 mM, it also doubled the K m (OAA) from 41 pM (•) to 89 iM (Fig. 3-12). Both reactions were stimulated by F-1,6-BP, but carboxylation was stimulated to a greater extent at subsaturating PEP concentrations, and the K m (PEP) was decreased three-fold in the presence of F-1,6-BP (Fig. 3-13). However, carboxylation at subsaturating levels of DIC was inhibited 27% by F-1,6-BP in an uncompetitive manner (Fig. 3-14). The photorespiratory compound glycolate inhibited carboxylation by less than 10% when PEP concentrations were below saturation. Decarboxylation was inhibited by substrates of the carboxylation reaction, PEP and DIC, but ADP appeared to stimulate decarboxylation at subsaturating ATP concentrations. The decarboxylation rate was decreased by 50% in the presence of 4 mM PEP, and the K m (OAA) was halved (Fig. 3-15), while 20 pM ADP decreased the K m (ATP) nearly three-fold (Fig. 3-16). In contrast, ATP inhibited

PAGE 110

97 Table 3-8. Influence of metabolites on PEPCK reactions. All reaction components were present at saturating concentrations for each reaction, except the effect of the metabolite on the reaction for a given substrate was measured at subsaturating concentrations of that particular substrate. Inhibition is reported vs. 1 mM substrate concentration for carboxylation and vs. 0.02 mM substrate for decarboxylation (unless noted differently). Metabolite Metabolite Concentration Degree of Stimulation or Inhibition Carboxylation PEP DIC ADP Decarboxylation OAA ATP mM Malate 4 Aspartate 4 DHAP 4 3 -PGA 4 F6P 4 F-1,6-BP 4 Glycolate 4 PEP 4 DIC 1 3 ADP 0.02 ATP 0.4 -9 -4 -6 14 34 -7 -4 -31 -61 -6 -27 -10 -7 -3 -2 -30 7 -37 -10 -22 21" -68" Note : A negative value indicates inhibition of the reaction. Substrate concentration was 0.004 mM. Substrate concentration was 0.05 mM.

PAGE 111

98 g Q d._uiuj ojd t _Buj HQVN \ouarf) c Q) m 2 a ffi r-l s o 13 o -H P O o a, H & 4-4 u H Q E m si P s 1H • +J m c 0) 10 4-> Q) n M tt) ft m 4-1 a) H P
PAGE 112

99 (J Q v-d.uiuu ojd t _6uu HQVN |Oiur/) 0) en 0) H a § a o +J IX X! H
M-l C CI) (1) Ul 0) (1) x; M H a ft • r< o ffi H n m A -P *— -rH o b —

PAGE 113

100 o o o o o o o o 00 CM o o o o o 6 o o d o d y D t-d.-Uioi ojd u _6uu HQVN \oiurf) -H 4-1 (fl u -P C i X O -Q U 1 •rl s tf ,-^ o IW -— •. -p -p o c
PAGE 114

101 0) w 0) u ft PL. O a .2 -p (S H >i X .0 o o p. H Ph H c fa i-d-UiuJ cud L _6uu HQVN IOlutY) q-l +> +> o c CD U) 4-1 Hi IH (1) H h ft 0. CN Li> H In n £ -P m •H u s ps & s-~~ •rH P* -—

PAGE 115

102 LU a u -3) ^.uiai ojd t _6w HQVN \ouirl) o a § -rH -P BJ 1 id u u CM w ft o 4-1 W (]> • £ •P -p C fl) c fj) (1) n ft ft CQ 1 ft ID PQ i 1 rH ^D 1 H fe H | fo Xi ^r -P -H m IS -u o C) • — a) m m -p W c ai m • (!) n u rH Ph n ft m (1) ] U ys 3 D1>H -H i En h

PAGE 116

103 ^(t-UjUJ ojd t _6uu HQVN loaiT/) O Q -p 0) 0} 0) ft ft m i I o c c -P
PAGE 117

104 < < ^jjilu OJd t _6Lu HQVN lowr?) 0) (0 0) u ft ft £ g c o X o u as o In "^

PAGE 118

105 h< 0} 0! Q) H ft 04 O c c o H -P H >, 8 td o 4 CM H 04 M O 04 i a Q) JC V a a ^{j.uiuj cud u _Buu HQVN puir/) -p -p o C (!) (U M-l 01 H 0) H u VU P^ vO o rH < n J3 -p (i) H u s 3 m 4^*. iH o [xj -—

PAGE 119

106 carboxylation by 68% at subsaturating levels of ADP. In the presence of 4 mM ATP, the K (ADP) for PEPCK carboxylation was doubled and the V mgx decreased by one-third (Fig. 3-17). Although the spectrophotometry method is a rapid and easy technique to use for most measurements of PEPCK, it does have its drawbacks. The PEPCK carboxylation spectrophotometry assay is coupled to MDH and NADH so that a change in absorbance at 340 nm wavelength occurs in a one to one ratio with the production of OAA. As soon as the carboxylation product OAA appears, it is reduced by MDH with NADH to yield malate. The decrease in absorbance is a direct measure of the oxidation of NADH and an indirect measure of OAA production. Because of this coupled assay method, the rate of carboxylation cannot be measured spectrophotometrically in the absence of MDH and NADH. The coupled assay method also precludes examining a direct effect of OAA on the carboxylation reaction, as any exogenously added OAA would be consumed by the MDH and NADH at a rapid rate, thus resulting in an artificially high oxidation rate of NADH. The 14 C method, although more timeconsuming and less flexible, does produce a direct measure of OAA formation. Using this technique, the presence of MDH and NADH in the reaction mixture was found to increase the reaction rate by six-fold (Table 3-9). Malate inhibited the reaction with or without MDH and NADH present by 25-30%, and this was a greater effect than observed with the spectrophotometric assay, where it resulted in only 10% inhibition (Table 3-8). The decarboxylation substrate ATP inhibited the reaction with MDH and NADH present by only half as much as the reaction measured when MDH and NADH were absent. Both of the radiochemical assays, with and without MDH and NADH present, were affected less than the spectrophotometric assay, which was inhibited 68% by ATP. The reaction in the presence of MDH and NADH was inhibited 33% by OAA, but in contrast, OAA appeared to stimulate the reaction by nearly three-fold when MDH and NADH were absent.

PAGE 120

107 a Q < Q) Cfl ffl y Pi H rti O o -H rH >< X o X! o o CM H Cm 8 M-l Cm a 3 E a) X! +-> B o Cm t _( t _U!UJ ojd t _6uj HQVN loujry) M-l -P •P c (U (1) M-l 0) M-l (1) H H ft • Cm rH i-l KJi ro X! -P a) •^ n :s p & ,*-s H o In **-*

PAGE 121

108 Table 3-9. Influence of various metabolites and MDH/NADH on the rate of PEPCK carboxylation measured as 14 CO ? uptake. Effector PEPCK Carboxylation Reaction Rate Without Stimulation Rate With Stimulation MDH/NADH or Inhibition MDH/NADH or Inhibiton jumol 14 C0 2 mg" 1 pro min~ 1 % pmol 14 C0 2 rag" 1 pro min" 1 % None 3.94 0.09 23.62 0.98 Malate 2.94 0.29 -25* 16.55 0.27 -30 ATP 2.87 0.05 -27 19.98 1.01 -15 OAA 11.22 0.30 285 15.71 0.14 -33 Note; A negative value indicates inhibition of the reaction.

PAGE 122

109 Discussion Earlier photosynthetic work with Udotea indicated that the alga has several C 4 ~like characteristics including photosynthesis that is largely Op-insensitive, a low photorespiration rate, low labeling of photorespiratory compounds, and C 4 acids that appear early and turn over rapidly rather than accumulating as end-products (122, 123). In an examination of enzymes capable of C 4 acid production, the only one that was found with activity comparable to that of Rubisco and the photosynthetic rate was PEPCK (123). Furthermore, in the presence of the PEPCK inhibitor MPA, photosynthesis of the Udotea. thallus exhibited C 3 -like characteristics, in that photosynthesis was inhibited by 2 the labeling of photorespiratory compounds increased, and the production of C 4 acids as early products was reduced (122, 123). These studies have provided good evidence that PEPCK functions in Udotea as a lightdependent carboxylase for photosynthesis. However, they do not indicate what drives the reaction in the carboxylation direction, given that in those higher plants where it has a photosynthetic role, the enzyme operates as a decarboxylase. This enzyme is found in a variety of organisms and is responsible for a number of quite different metabolic functions. In Udotea, PEPCK appears to be similar to the E.C. 4.1.1.49 enzyme as found in higher plants, yeast, bacteria and parasitic protozoa (33, 147, 150) because the enzyme utilizes the adenine nucleotide. Furthermore, it is an oligomer (Reiskind et al unpublished data). This contrasts with the E.C. 4.1.1.32 enzyme found in vertebrates, Euglena, and helminths, which uses the guanine or inosine nucleotide for its reaction and is a monomer (116, 117, 130). A difference between the two forms of the PEPCK enzyme is indicated by the monomeric PEPCK enzymes' sharing a 62-64% amino acid sequence similarity, but no significant similarities to the yeast gene sequence and the sequences from the corresponding enzymes of rat liver

PAGE 123

110 or chicken (50). Thus, though the two enzymes catalyze essentially the same reaction, the two proteins are quite dissimilar. Carboxylation by the Udotea PEPCK enzyme is similar to that of the higher plant Panicum which has a pH optimum of 6.8 and a K m (PEP) of 1.46 mM (151). The Udotea K m (C0 2 ) is similar to that from Urochloa, at 2 5 mM (74). In this investigation of Udotea, MPA inhibited the carboxylation activity of the purified enzyme in a competitive manner. The PEPCK from Panicum maximum is also inhibited by MPA (120), as is the enzyme from Urochloa, but here the inhibition is noncompetitive with regard to OAA (33). The K (ADP) also was comparable to the cytoplasmic PEPCK of the brown alga Ascophyllum (78). Udotea PEPCK interaction with other substrates is also similar to the enzyme from Phaeodactylum which has a K m (PEP) and (ADP) of 2 2 and 0.048 mM, respectively, at the optimum pH of 6.2 (72). The K m (HC0 3 ") for Udotea compares with that of PEPCK from the diatom Phaeodactylum (72) and the helminth Ascaris (116). It is also similar to that for both the autotrophic and heterotrophic forms of Euglena enzyme (117), although that enzyme uses GDP rather than ADP. The K (ADP) also was comparable to that from the PEPCK of the cytoplasm of chicken liver (130). For decarboxylation, the K (OAA) was the same as that determined for Chloris at 0.035 mM (67) and similar to Panicum at 0.025 mM (33) and Trypanosoma at 0.027 mM (150). However, the KJATP) of 2 uM. for the Udotea enzyme is approximately ten-fold lower than any values reported in the literature for the higher plant enzyme (33, 67, 151). The enzyme from Udotea is not unique among PEPCK enzymes. It shares kinetic characteristics with PEPCK enzymes from C 4 plants, unicellular algae, brown algae, a protozoan, and a roundworm. Among these organisms, PEPCK functions as a carboxylase in some and a decarboxylase in others. The question to be answered is how this enzyme plays a role in the C^-like photosynthetic metabolism observed in Udotea.

PAGE 124

Ill This examination of the interaction of Udotea PEPCK with its substrates indicates that the enzyme has a greater affinity for the substrates of the decarboxylation reaction, OAA and ATP, than for the carboxylation substrates. If this were the only basis for consideration, it might be reasonable to conclude that Udotea PEPCK functions predominantly as a decarboxylase. In this study, MPA inhibited PEPCK decarboxylation in a competitive manner with respect to OAA. In contrast, in mammalian systems, MPA inhibition of PEPCK is noncompetitive with respect to the GTP-dependent decarboxylation of OAA, which halts gluconeogenesis (80). Earlier enzyme investigations in Udotea showed that the only carboxylase/decarboxylase enzyme normally associated with C^ photosynthesis which had substantial activity in the alga was PEPCK (123). In the terrestrial C 4 -PEPCK plants, the initial carboxylation is carried out by PEPC with the subsequent decarboxylation step catalyzed by PEPCK. However, PEPC activity in Udotea extracts was minimal (123). There are three major differences between Udotea PEPCK and the PEPC of higher plants: PEPCK uses C0 2 as its inorganic carbon substrate while PEPC uses HC0 3 ~ (4), the metal ion cof actor needed by PEPCK is Mn while PEPC requires Mg 2+ (4), and PEPCK carboxylation activity requires ADP while PEPC does not (4). An attempt to detect carboxylation activity in Udotea crude extract with only Mg 2+ as the metal ion or in the absence of ADP did not result in any OAA production (123). Thus the evidence strongly indicated that the enzyme from the extract operating in carboxylation was PEPCK and not PEPC. However, for C^ photosynthesis to operate, there must be an initial carboxylation reaction followed by a decarboxylation which provides a higher concentration of C0 2 for Rubisco in the chloroplast. In the absence of good evidence for other decarboxylase enzymes, it was hypothesized that perhaps PEPCK operated in Udotea in both the carboxylating and decarboxylating directions. For this to occur without futile metabolite and energy cycling, there would

PAGE 125

112 have to be a separation of the carboxylation and decarboxylation reactions. The working hypothesis was that one isozyme of PEPCK might be operating in the cytosol as a carboxylase while a second isozyme, located in the chloroplast, might operate in the decarboxylating mode to provide the C0 2 for Rubisco (.122, 123). In terrestrial C, plants, the separation of carboxylation and decarboxylation functions is achieved through Kranz anatomy (48), but in CAM species, the separation of carboxylation and decarboxylation is temporal, between day and night, rather than spatial (112), while in low photorespiration-state Hydrllla verticallata, intracellular compartment at ion of enzymes sparates carboxylation from decarboxylation (121). Thus, Kranz anatomy is not always essential for a plant to be able to utilize a form of C^-metabolism for photosynthesis. During the isolation of PEPCK from Udotea, only one activity peak was detected in the eluant from the weak anion exchange chromatography column, and this was consistently found in a number of preparations. In organisms where two isozymes are present, the two forms can usually be distinguished by ion exchange chromatography (58, 69). In addition, cellular fractionation of Udotea resulted in the detection of PEPCK in only the cytosolic fraction and not the chloroplast fraction (Jun and Reiskind, unpublished data). Thus, it appears there is only one isozyme of PEPCK in Udotea, located in the cytosol, which functions predominately as a carboxylase. These data indicated that the working hypothesis required modification to consider that PEPCK may be functioning only as a carboxylase, with the subsequent decarboxylation performed by another enzyme, such as NAD-malic enzyme, which is also present in Udotea (123). In the higher plants, the ATP-PEPCK functions in the species of one of the C 4 photosynthetic subgroups as a decarboxylase in the cytosol of bundle sheath cells to provide COp for Rubisco in the chloroplasts of these cells (48). The enzyme is operating essentially to increase the

PAGE 126

113 concentration of COp in the vicinity of Rubisco so as to favor carboxylation over oxygenation with a concomitant decrease in the PCO cycle. In some of the brown algae, PEPCK operates in a lightindependent carboxylating mode to provide the carbon skeletons for anapleurosis (78). In yeast, PEPCK functions to supply the cells with glucose from threeand four-carbon precursors (36). High activity of PEPCK is also found in Ascaris, a parasitic worm, where the enzyme operates as a glycolytic Confixing enzyme (116). In the unicellular alga Euglena grown under heterotrophic conditions, GDP-PEPCK plays a role in anaerobic C0 2 fixation for synthesizing odd-numbered fatty acids via the methylmalonyl-CoA pathway (117). PEPCK is found in mammals as well, where it functions in replenishing depleted glucose levels in the blood through gluconeogenesis with the enzyme operating in the decarboxylating direction in humans (69), rats (31), and chickens (43). Thus, PEPCK is involved in a number of different metabolic functions ranging from producing cellular energy in the form of ATP for marine diatoms in low light (72), to producing GTP during carboxylation for fatty acid synthesis (117) and glucose in gluconeogenesis (152), as well as providing a source of C0 2 for an enzyme in the next step of metabolism. Given this wide variety of metabolic functional roles, this investigation was carried out to determine the kinetic features of Udotea PEPCK to ascertain how this enzyme may play a role in the C 4 ~like photosynthetic metabolism of this marine alga. The pH profiles for carboxylation and decarboxylation suggested that the pH of the environment to which the enzyme was exposed in vivo could play a role in determining the predominant direction of the reaction. If the cytosolic pH were in the range of 6.5 to 7.0, the carboxylation reaction could run up to two to three times more rapidly than decarboxylation. Even at a pH of 7.0, carboxylation proceeded at a rate 1.5-fold greater than decarboxylation. Assuming that the cytosol,

PAGE 127

114 where PEPCK is believed to be located, has a pH close to neutral (47), then carboxylation by PEPCK would be favored. Increasing the pH from 6.8 to 7.8 increased the affinity for C0 2 by nearly four-fold as the K (CO.,) of Udotea PEPCK decreased with increasing pH. A similar situation has been reported for the diatom Phaeodactylum trlcornutum (72), where the K m (C0 2 ) decreased eight-fold over the pH range 6.2 to pH 7.6 (72). In the diatom system, the PEPCK is also postulated to function as a carboxylase. However, this must be weighed against the decrease in the V of carboxylation rate as the pH rises above the optimum. An examination of the inorganic carbon source for carboxylation indicated that Udotea PEPCK utilized free C0 2 as has been found for PEPCK in organisms as diverse as the higher plant Urochloa (74), the brown alga Ascophyllum (78), a bacterium (44), and a parasitic worm (150). However, the enzyme from Phaeodactylum, with which Udotea PEPCK shares several characteristics, is reported to use HCOj~ as the substrate (72). Because of its use of C0 2 rather than HC0 3 ~ as substrate, the possibility was considered that 2 might act as a competitive inhibitor, in a manner reminiscent of the situation with Rubisco (26). The other major carboxylating enzyme, PEPC, is not sensitive to inhibition by 2 (26), but its inorganic carbon source is HCO~ (4). The present study indicated that carboxylation by Udotea PEPCK was not inhibited by 2 even at 100% 2 (gas phase) and subsaturating DIC concentrations, where a competitive effect would be maximized. This is, therefore, further evidence that PEPCK could play a similar role to PEPC in terrestrial C, plants which provides the initial C0 2 fixation step unhampered by inhibitory effects of dissolved 2 in the vicinity. An examination of the effect of OAA on the PEPCK carboxylation reaction using the 1/f C method indicated that OAA appeared to stimulate carboxylation (Table 3-9), which seemed an unlikely occurrence. The

PAGE 128

115 data can be explained by considering that a 14 C0 2 -OAA exchange reaction, also catalyzed by PEPCK, was occurring in the reaction mixture. The OAA added exogenously was becoming radioactive due to the exchange reaction, and this was in addition to the labeled OAA produced by the carboxylation reaction. Therefore, there were two reactions producing 1z, C-OAA in the reaction vial. This exchange reaction proceeds 30 times faster than the carboxylation reaction and has been documented for PEPCK from pig liver (152) and chicken embryo liver (130). However, when the 14 C carboxylation reaction was performed in the presence of MDH and NADH, OAA inhibited the reaction. This result could come about either because the OAA was truly inhibiting the PEPCK carboxylation reaction or because the OAA was inhibiting the MDH catalyzed reduction of OAA. However, because the MDH reaction was operating at a capacity far in excess of PEPCK carboxylation, the former seems to be the more likely explanation, and this would be in accord with a build-up of product reducing the PEPCK carboxylation rate. A question arises as to whether the exchange reaction was being measured in the spectrophotometric method for carboxylation instead of the true carboxylation reaction. The exchange reaction would not be measurable by the oxidation of NADH, which is used as a measure of the carboxylation rate in the spectrophotometer. Also, in the reaction mixture for that assay, the only OAA present was that produced as a result of the carboxylation of PEP. Because there is an abundance of MDH activity present, as soon as the OAA is produced, it can be reduced by the MDH with NADH to malate. To understand further how PEPCK may be functioning In vivo, the effects of cellular metabolites on the enzyme activity were examined. The C, acid malate was an uncompetitive inhibitor of Udotea PEPCK, causing a slight decrease in the apparent K m (DIC) and lowering the V max In Phaeodactylum the C 4 acid aspartate had the opposite effect by increasing the reaction velocity (72). However, in other organisms, C 4

PAGE 129

116 acids have been reported not to affect either the carboxylation or the decarboxylation reaction (72). Of the phosphorylated compounds examined, ATP exerted the greatest effect on the Udotea carboxylation reaction with an inhibition of 68%. The inhibition was partially competitive with respect to ADP, and ATP lowered the V and increased the apparent K (ADP) Competitive max *in inhibition by ATP of the carboxylation reaction has been reported in the brown alga Laminarla, which catalyzes a lightindependent carboxylation (159), in the liver fluke Fasciola with respect to PEP (88), and in Ascaris with respect to PEP and IDP (116). ATP also inhibited carboxylation by PEPCK in yeast by 43% (147). In regard to decarboxylation, reports of inhibition by ADP are fewer than for ATP in carboxylation, but in the higher plant Panicum, ADP is a competitive inhibitor of the decarboxylation reaction with respect to ATP (151), while in Urochloa, the combination of ADP and Mn + functions as a noncompetitive inhibitor with respect to ATP (33). In Udotea, ADP appeared to stimulate decarboxylation, but this could be attributed to the interaction of ADP with pyruvate kinase in the reaction mixture to increase the concentration of ATP for the reaction, rather than actually stimulating the decarboxylation reaction directly. Related to the substrate function of ADP for PEPCK carboxylation is its interaction with Mn 2+ and the enzyme. Several inhibitor studies have indicated that the binding of ADPMn" to the active site of the enzyme protects the active thiols in the active site of yeast PEPCK against deactivation (36, 50). In addition to binding with the nucleotides, free Mn 2+ must bind to a site on PEPCK to effect catalysis (33, 67, 152). For most enzymes, this Mn 2+ is essential and cannot be replaced by any other divalent metal cation, including Mg 2+ (33). In Udotea, the presence of Mg 2+ along with Mn 2+ inhibited the carboxylation reaction. The decarboxylation reaction in Urochloa is also inhibited by Mg + under similar conditions (33).

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117 The phosphorylated organic acid 3-PGA inhibited decarboxylation by at least 50% in the enzyme from the higher plants Chlorls (67) and Urochloa (33) and in the diatom Phaeodactylum, where the enzyme operates as a carboxylase (72). Likewise, 3-PGA inhibited carboxylation in Udotea by 61% in a noncompetitive manner. However, in an anaerobic bacterium of the human mouth, Veillonella, 3-PGA actually activates PEPCK for gluconeogenesis to produce PEP during lactate metabolism (38). Several other phosphorylated compounds influenced the decarboxylation reactions of the higher plant species. Chlorls enzyme is inhibited by DHAP, F6P, and F-1,6-BP (67). Urochloa PEPCK is inhibited by F6P and F1,6-BP, both against free Mn 2+ (33). PEP functions as a noncompetitive inhibitor versus OAA for PEPCK decarboxylation in Panlcum (151). In Udotea, both DHAP and F-1,6-BP inhibited carboxylation by approximately 30% with regard to DIC. However, F-1,6-BP stimulated carboxylation by 34% and decreased the apparent K m (PEP) from 1.55 to 0.54 mM. Fructose6P, on the other hand, slightly stimulated carboxylation while inhibiting decarboxylation versus OAA. At physiological concentrations, PEP and DIC inhibited decarboxylation, but PEP also halved the apparent K (OAA), from 0.04 mM to 0.02 mM. Decarboxylation was inhibited by DIC (data not shown), as also indicated for the interaction of C0 2 with the enzyme from higher plants (4). One of the major metabolic pathways operating in the cytosol, where PEPCK appears to be located in Udotea, is glycolysis. However, PEPCK is hypothesized to be functioning in photosynthetic carbon assimilation, so the intracellular milieu at that time must be considered. As the function of glycolysis is to provide pyruvate to enter the tricarboxylic acid cycle for the subseguent generation of ATP, it is reasonable to assume that glycolysis is functioning at a minimal level because sufficient ATP is provided by the light reactions of photosynthesis. One of the control enzymes in glycolysis is phosphofructokinase, which is inhibited by ATP (148). Glycolysis does

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118 occur in plant tissue, but its need for operation during photosynthesis is probably diminished. A major function of PEPCK in other organisms is in gluconeogenesis, where the pathway remobilizes glucose. As is the case for glycolysis, there is little need for gluconeogenesis to be functioning in actively photosynthesizing cells. Gluconeogenesis does occur in plants, both in non-photosynthetic tissue for the breakdown and remobilization of stored fats (148) and in the dark in all plant cells. Thus, a major source of cytosolic metabolites that could interact with PEPCK would be the chloroplast during photosynthesis. The DHAP transported out of the chloroplast can be isomerized in the cytosol to 3-phosphoglyceraldehyde (3-PGAl). The combination of DHAP and 3-PGA1 produces the 6-carbon sugars. Since F6P and DIC inhibit the decarboxylation reaction while F-1,6-BP enhances it at subsaturating PEP concentrations, it may be that the metabolites in the cytosol during photosynthesis alter the kinetics of the reaction enough so that carboxylation can occur at an appreciable rate. After OAA is produced in the carboxylation reaction, the high activity of cytosolic MDH, which has a high affinity for OAA with a K (OAA) of 0.06 mM (Rattray and Bowes, unpublished), may function to rapidly convert the OAA to malate. The removal of OAA may aid in pulling the reaction in the carboxylation direction. In the blood fluke Leishmanla, high PEPCK activity is also accompanied by high MDH activity (105). In this organism, where PEPCK functions as a carboxylase, the high MDH activity may work to bring about succinate production in conjunction with fumarate reductase. Thus, here is an example of PEPCK and MDH working in concert to accomplish the metabolic task. The situation appears to be similar in Udotea, though the metabolic role is different. Thus, although Udotea PEPCK appears to have a higher affinity for the substrates of the decarboxylation reaction, it is hypothesized that the microenvironment in the cytosol, where PEPCK is located, is such

PAGE 132

119 that PEPCK is capable of carrying out the carboxylation of PEP to the extent that the subsequent metabolism of the OAA produced is able to provide a higher C0 2 concentration for Rubisco in the chloroplast. The cytosolic pH of approximately 7.2 (47) coupled with the high MDH activity facilitates the operation of PEPCK in Udotea in the carboxylating direction. Enhancing the carboxylation of RuBP relative to its oxygenation is apparent in Udotea from the low C0 2 compensation point, low photorespiration rate, low concentrations of photorespiratory compounds, and low sensitivity of photosynthesis to 2 as well as the early appearance of C, acids which rapidly turn over (123). The important role of PEPCK in initiating this metabolism was clearly demonstrated when Udotea thalli were treated with MPA. When PEPCK was inhibited, the photosynthetic characteristics of the alga were very Cjlike: photosynthesis became 2 -sensitive, an increase in photorespiration occurred as evidenced by an increase in the C-labeled photorespiratory intermediates detected, and the early production and subsequent turnover of C, acids disappeared (123). The current working hypothesis of the photosynthetic metabolism in Udotea is presented in Fig. 3-18. According to this scheme, C0 2 would be fixed by PEPCK in the cytosol to OAA, which is then reduced by MDH and NADH to malate. Not only would this step stabilize the C 4 acid, it would also serve as a mechanism for re-oxidizing NADH as also suggested for Chlorls (66). The malate produced in the cytosol would be transported to the chloroplast where the decarboxylation event would occur by an enzyme that remains to be determined. One possible enzyme that could catalyze this reaction is NAD-malic enzyme. In terrestrial C, species, this enzyme is located in the mitochondria of bundle sheath cells, but in the marine diatom Phaeodactylum, this enzyme is reportedly present in the chloroplasts (72). If NAD-malic enzyme also be present in the chloroplasts of Udotea, the C0 2 released could be fixed into the PCR cycle by Rubisco, while the pyruvate could be used to regenerate PEP

PAGE 133

en U Di o -* o rH 120 C O •H D 3 ft >i X! 0) U c o -rH -P (d K rH D O X! u cd O m •P 0) c 0) Di-P a) ft 0) o o id u 0) x: +J o G id <8 •w XI ts E3 d) •P id o m O -P o o O Q) ffl d) s o CD -p >1 rH o S d) X! H +J o -P ft-P Q) O .* U U td (d X! C o A U rH cd T3 td c x; -p o o m •-H X! 3 C CC (d o i (U TS CO o O 3 -P TS O U C O H -P CQ 0) •H CD Cd rue ft ft cd O -M rlUM wood) Xi en .0) s-i td H o o d) en CD O O -P ft >1 o o ffj o ttl • x: -p c -H C o -rH -P -P rd > U >i ft C >i td X! 00 C H -rH I w n U O Ph (llftH HHft Oi 0) -H >iXl h X! -P --P U TS CO td m td 0) 4-1 rH n td ft o X o

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121 via pyruvate Pdikinase. A sufficient activity of this enzyme to support the photosynthetic rate has been found in Udotea (123). In this way, then, Udotea would be able to provide Rubisco with an elevated concentration of C0 2 i.e., a co 2 -concentrating mechanism, which accounts for the C 4 -like photosynthetic features displayed by this alga.

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BIOGRAPHICAL SKETCH Beth Ann Lilly was born in Reading, Pennsylvania, on October 1, 1963. She graduated from Governor Mifflin Senior High School in Shillington, Pennsylvania, in June 1981, matriculated at Millersville University in Millersville, Pennsylvania, as a biology major in August 1981 and graduated with a Bachelor of Science cum laude in August 1985. She then moved to Gainesville, Florida to commence graduate studies in botany under the direction of Dr. George Bowes at the University of Florida in August 1985. Beth was awarded the Master of Science degree in August 1988. She immediately began research toward the doctorate in botany, under the direction of Dr. George Bowes. In August 1992, she married Gregory Burch in Gainesville, Florida. Beth became an assistant professor of biology at Huntington College in Huntington, Indiana in September 1993 and graduated with the degree Doctor of Philosophy in December 1993. 135

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I certify that I conforms to acceptable adequate, in scope and Doctor of Philosophy. have read this study and that in my opinion it standards of scholarly presentation and is fully quality, as a dissertation for the degree of WS^-d I certify that I conforms to acceptable adequate, in scope and Doctor of Philosophy. Bqs&es, Chairman Professor of Botany have read this study and that in my opinion it standards of scholarly presentation and is fully quality, as a dissertation for the degree of I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a -'dissertation for phe degr.ee of Doctor of Philosophy. J \ / 1/ C^CT C V /[ Richard C. Smith' "^" Professor of Botany I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. les M. Allen, Jr. // Charl Professor of Biochemistry 2 Biology Zi Molecular I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. lafles "L '"-tSjay / Charl— Associate Profe,ers] Science f Horticultural This dissertation was submitted to the Graduate Faculty of the Department of Botany in the College of Liberal Arts and Sciences and to the Graduate School and was accepted as partial fulfillment of the requirements for the degree of Doctor of Philosophy. December 1993 Dean, Graduate School


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