Citation
Acid-soluble phosphate compounds of corn roots

Material Information

Title:
Acid-soluble phosphate compounds of corn roots
Creator:
Wise, Byron Hooper, 1925-
Place of Publication:
1962
Publisher:
[s.n.]
Language:
English
Physical Description:
vii, 58 leaves : ill. ; 28 cm.

Subjects

Subjects / Keywords:
Chromatography ( jstor )
Elution ( jstor )
Formates ( jstor )
Hexoses ( jstor )
Ions ( jstor )
Nucleotides ( jstor )
Phosphates ( jstor )
Phosphorus ( jstor )
Resins ( jstor )
Root tips ( jstor )
Botany thesis Ph. D
Corn ( lcsh )
Dissertations, Academic -- Botany -- UF
Phosphorus compounds ( lcsh )
Roots (Botany) ( lcsh )
Genre:
bibliography ( marcgt )
non-fiction ( marcgt )

Notes

Thesis:
Thesis (Ph. D.)--University of Florida, 1962.
Bibliography:
Includes bibliographical references (leaves 52-57).
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Byron Hooper Wise.

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Source Institution:
University of Florida
Holding Location:
University of Florida
Rights Management:
This item is presumed in the public domain according to the terms of the Retrospective Dissertation Scanning (RDS) policy, which may be viewed at http://ufdc.ufl.edu/AA00007596/00001. The University of Florida George A. Smathers Libraries respect the intellectual property rights of others and do not claim any copyright interest in this item. Users of this work have responsibility for determining copyright status prior to reusing, publishing or reproducing this item for purposes other than what is allowed by fair use or other copyright exemptions. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder. The Smathers Libraries would like to learn more about this item and invite individuals or organizations to contact the RDS coordinator(ufdissertations@uflib.ufl.edu) with any additional information they can provide.
Resource Identifier:
030455564 ( ALEPH )
36844503 ( OCLC )

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ACID-SOLUBLE PHOSPHATE COMPOUNDS

OF CORN ROOTS













By

BYRON HOOPER WISE


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












UNIVERSITY OF FLORIDA
December, 1962
















ACKNOWLEDGEMET


The writer expresses his appreciation to Dr. T. E. Humphreys for his thoughtful guidance during the course of this research; to Dr. G. R. Noggle for encouragement and financial support through the Department of Botany during the entire doctoral program; to Dr. A. T. Wallace for encouraging the writer to undertake the program; to Drs. R. D. Powell and T. W. Stearns for serving on the committee.

Special credit is due my wife, Winnie Sue Wise, who made this work possible.


ii














GIOSSMA


P: phosphorus R5P: ribose-5-phosphate

PP: pyrophosphate GlP: glucose-l-phosphate

I: inorganic phosphorus G6P: glucose-6-phosphate

TP: total phosphorus FiP: fructose-l-phosphate

G3P: glyceraldehyde-3-phosphate 76P: fructose-6-phosphate

MPW: monophosphoglycerate FDP: fructose-i1,6-diphosphate

2PGA: 2-phosphoglycerate HMP: hezose monophosphate

3PGA: 3-phosphoglycerate HOP: hexose diphosphate

WO: 2,3-diphosphoglycerate

MT or DPMH: oxidized or reduced diphosphopyridine nucleotide TPN or TPIE: oxidized or reduced triphosphopyridine nucleotide AMP, ADP, ATP: adanosine mnao-, di-, triphosphates CP, CDP, CTP: cytidine mnmo-, dij triphosphates GM, GDP, GTP: guanosine mono-, di-, triphosphates UWP, UMP, UTP: uridine _.no-, di-, triphosphates

IW, ITP: inosine di-, triphosphates UPPG: uridine diphosphate glucose


iii





..- w.


TABLE OF CO1TETS


Page


ACKNOWLEDUMEUT . . . . . . . GIOSSARY . . . . . . . . . .

LIST OF TABLE ....... LIST OF FIGURES . . . . . . . STATEMENT OF PROBLEM . . . . REVIEW OF LITERATURE . . . . MATERIALS AND MEDS . . . .

Plant Materials


. . . . . . . . . . ii

. . . . . . . . . . iii

.......... vi

. . . . . . . . . . vii

. . . . . . . . . . 1

. . . . . . . . . . 2

. . . . . . . . . . 16


Preparation of Extracts

Preparation of Ion Exchange Colums

Column Chromtography

Analytical Methods

Phosphorus (TP, IP, 7-min P) assay Assays for sugar phosphates Assay for glycerates Enzymatic assays for phosphorus compounds
G6P and F6P
Uridine diphosphate glucose (UPG) Microdetermination of ATP with firefly lantern extract Analysis from UV absorbency measurements


iv









Page

RESUITS . . . . . . . . . . .... . . . . . . . . . . 26

Dovmx 1-Chloride Chromatography of Known Compounds Douex 1-Formate Chromtography of Known Compounds

Dovex 1-Chloride Chromtogrphy of Corn Root Tip Extracts

Dowex l-Formste Chromtography of Eluates from Dovex 1-Chloride Separations of Corn Root Tip Extracts

Direct Dowex 1-Formate Chromatography of Corn Root Tip
Extracts

ATP Assay of Corn Root Tip Extracts Phosphorus Compounds Identified in the Root of the Etiolated Corn Seedling

DISCUSSION . . . . . . . . . . . . . . . . . . . . . . . . 49

SMAR . . . . . . . .... . . - . . . . . . . . . . . . 51

:JOGRAPHY . . . . . . . . . . . . . . . . . . . . . . 52

MOGRAPHICAL SE3'E . . . . . . . . . . . . . . . . . . . 58



























v


I- - - - -






1w


LIST OF TABLE

Table Page

1. PHOSPHORUS COTENT OF A TRICHOROACETIC ACID EXTRACT
OF 63.2 GRAms OF CORN ROOT TIPS, BEFORE AND AyTER
TREATMENT WITH DOWEX 50 . . . . . . . . . . . . . 29

2. PHOSPHO1US CONTT OF SEVERAL TRICHIDROACETIC ACID
EXTRACTS OF CORN ROOT TIPS, AFTER TREATMENT WITH
DOWEX 50 . . . . . . . . . . . . . . . . . . . . 30

3. TP RECOVERY IN THE DOWEX 1-FORMATE CHROMATOGRAPHY OF
THE 0.01 N HC1 SECTION FROM DOWEX 1-CHWRIDE
CHROMATOGRAPHY OF FOUR CORN ROOT TIP EXTRACTS . . . 34

4. ABSORBANCY RATIOS AT 250, 260, 280 mu . . . . . . . . 40

5. PHOSPHATE COMPOUND OF CORN ROOT TIPS . . . . . . . . 48


vi















LIST OF FIGURES

Figure Page

1-4 DOWEX l-CHI)RIDE CHROMATOGRAPHY OF TRICHLOROACETIC
ACID EXTRACTS OF CORN ROOT TIPS . . . . . . . . 31

5 DOWEX 1-FORMATE CHROMATOGRAPHY OF THE WEAK ACID
PHOSPHATE COMPOUNIE FROM A TRICIDROACETIC ACID
EXTRACT OF 18.6 GRAMS OF CORN ROOT TIPS . . . . . 32

6 DOWEX 1-FORMATE CHROMATOGRAPHY OF THE SUGAR NDNOPHOSPHATEs (PEAK 1) FROM AN EXTRACT OF 38.3 GRAME OF
CORN ROOT TIPS . . . . . . . . - . . . . - . . 36

7 DOWEX 1-FORMATE CHROMATOGRAPHY OF THE 0.02 N HCl
SECTION OF A TRICHIDROACETIC ACID EXTRACT OF
57.1 GRAMS OF CORN ROOT TIPS . . . . . . . . . . . 38

8 DIRECT DOWEX 1-FORMATE CHROMATOGRAPHY OF A TRICHIDROACETIC ACID EXTRACT OF 51.o GRAMS OF CORN
ROOT TIPS. . . . . . . . . . . . . . . . . . . . . 41

9 DIRECT DOWEX 1-FORMATE CHROMATOGRAPHY OF A TRICHIOROACETIC ACID EXTRACT OF 51.8 GRADE OF CORN ROOT
TIPS . . . . . . . . . . . . . . . . . . . . . . . 42

10 DIRECT DOWEX 1-FORMATE CHROMATOGRAPHY OF A TRICHIOROACETIC ACID EXTRACT OF 71.9 GRADE OF CORN ROOT
TIPS . . . . . . . . . . . . . . . . . . . . . . . 43

11 ULTRAVIOLET SPECTRA OF THE POOLED TP PEAKS FROM
DIRECT DOWEX 1-FORMATE CHROMATOGRAPHY OF
TRICHLOROACETIC ACID EXTRACTS OF CORN ROOTS. . . . 46


vii


- njpprmw -7














STATEMT OF THE PROBLEM


The glycolytic sequence represents a crucial segment in the

intermediary metabolism of the plant and animal cell. The operation of the glycolytic system can be expected to be influenced by the levels of each substrate and by the activity of each enzyme, in the series.

The presence in higher plants of most of the glycolytic intermediates is documented. However, very little is known about either the concentrations of these compounds, and their associated coenzymes, in normal plants, or their changes in relation to physiological changes. The reasons for this paucity of information can be traced to the severe technical difficulties encountered by various workers in applying quantitative analytical methods to plant materials, particularly the higher plants.

The object of this investigation is to develop methods to determine the levels of various glycolytic intermediates in plant tissue etiolatedd corn seedling roots) using ion exchange colunm chromatography as the basic means of separation.






11















REVIEW OF LITERATURE


The mthods employed in this investigation are based on the

separation of acid-soluble, phosphorus-containing compounds of plant tissue prior to their quantitative determination by various methods. The study involves a broad general survey of methods, including phosphorus assay, assay of sugar phosphates, assay of associated coenzymes, extraction methods, various types of ion exchange chromatography, and, finally, a review of the methods which have been used to study phosphorus compounds in plants. The following review is presented in the above sequence. It will be noted that most of the work in the field has been done with animal tissues. It will also be noted that there is some overlapping in presenting the pertinent articles. This occurs because it was thought that a review stressing methods would be more useful if the recent work involving ion exchange chromatography be presented with a modicum of detail.

Many methods have been described to assay phosphorus in biological material. Most procedures depend on the intense blue color which is produced ihpn the phosphomolybdate complex is reduced by various agents. The color intensity is measured in a photoelectric colorimeter ageinst a standard series of known phosphate concentrations.

One of the earliest methods (15) utilized hydroquinone for

reduction; however, the color vas evanescent. Fiske and SubbaRow


2


,~





3


(30) discussed several factors affecting accurate phosphorus determinations. Variations of their method, involving reduction by aminonaphtholulfonic acid in the presence of 128014, are still popular

(13). The method is reportedly insensitive to a 30% variation in acid, molybdate, or reducing agent concentration. Berenblum and Chain (17) described a method in which the phosphomolybdic acid is reduced to the blue comqlex by shaking with a mixture of butyl alcohol and stannous chloride and separating the blue alcoholic layer. The method is said to be insensitive to ten-fold increases of the reagents. The Gomori

(31) modification uses the photographic developer Elon (methyl-p-aminophenol sulfate) for reduction.

In general, IP* is determined by measurement of color production after 10 to 15 min of development; TP is similarly determined after wet-ashing the sample in K2S04.

The estimation of IP in the presence of ATP with a minimum of

molybdate-catalyzed hydrolysis of ATP is described by Marsh (54). The excess molybdate is removed as a citrate complex after the extraction of phosphomolybdate by butanol. Alternately, the IP can be precipitated by CaCl2-Ca((K)2 to remove various labile phosphate enters, such as creatine phosphate and ATP.

Several colorimetric methods used for estimation of sugars and sugar phosphates are described by Ashwell (lo). The enthrone, cysteine-carbazole, and orcinol procedures (10) are colorimetric assays for hexose, fructose, pentose, and their phosphate derivatives. Two compounds as similar as ribose-3-phosphate and R5P my be differentiated under carefully controlled heating conditions (T). In


See glossary.









practice, the results have been equivocal (10) except here considerable purification wa attained prior to colorimetry. However, Bartlett (13) has reported the determination of ketohexose phosphate vith cysteine-carbazole, and pentose phosphate vith orcinol, in fractions containing a mixture of these esters. Further, Bartlett has claimed quantitative assay of G6P by subtracting the cysteine-carbazole result from the enthrone result obtained on assaying a mixture of G6P and F6P. However, Helbert and Brom (35) found that color production in the anthrone assay varied from hexose to hexose. GlP has been estimated in mixtures of hexose phosphates by measuring the increase in IP after 7 min at 100 C in 1 N HC (45).

Enzymatic methods are available for the estimation of sugar phosphates. FD? can be assayed by the method of Slater (70), using the rabbit muscle fraction A of Racker (63), vhich contains aldolase, triosephosphate isomerase and d-glycerol phosphate dehydrogenase. The change in optical density at 340 mu due to oxidation of added DPNH is used to calculate FDP. The method measures the sum of FOP, G3P, and MEAP.

F6P can also be estimated by Slater's procedure (70). When Racker's (63) rabbit muscle fraction B (containing phosphohexokinase) and ATP are incorporated in the above reaction, FDP and F6P are measured. F6P is measured by the difference between this method and that for FDP alone, in the absence of G6P.

G6P can be assayed by the method of Ochoa et al. (60) by measuring the change in OD due to reduction of TPW by glucose-6-phosphate dehydrosenase. If phosphohexose isomerase is added to the system, F6P may be estimated by difference (70). Greengard 2t L1. (33) estimated G6P,






5


according to the procedure of Slater (70), by measuring the chang of fluorescence of TPNH, and, alternatively, measured ATP by adding glucose and hexokinase to the reaction mixture used for the estimation of G6P. Kornberg (4o) determined ATP by this method (spectrophotometrically). Ochoa et al. (60) assayed pyruvate spectrophotometrically in the presence of lactic dehydrogenase and DMH.

Seraydarian et .1. (69) adapted fluorometric methods to the determination in muscle extracts of millimicromolar quantities of

(a) FDP plus triose phosphates and (b) hexose monophosphates. The two groups wre not separated; the authors felt that the two groups wre important in representing glycolytic intermediates of similar phosphate composition.

I The levels of adenine nucleotides, especially ATP, are particularly important in biochemistry of animal and plant tissues. ATP has been estimated by the method of Slater (70) and the micro method of Seraydarian (69). McElroy's finding (47) that added ATP caused light production in the darkened extracts of firefly lanterns opened the possibility of using the phenomenon to assay ATP. Further experiments by the McElroy group (48) showed that the production of light in firefly extracts (FFE) depends on a heat-labile enzyme (luciferase), a heat-stable, yellow-green luminescent compound (luciferin), an inorgnic ion (Mg , Co', or 16), oxygen, and ATP. A partial purification of the FFE (49) resulted in the removal of myokinase and adenosinetriphosphatase, and revealed the specificity of the luciferin-luciferase reaction to ATP. Strehler and Totter (71) described the application of the system to the estimation of mixtures of ADP and ATP.






6


The FFE assay for ATP has been used in investigations on

aninmi tissues, especially the nervous system. Single frog sciatic nerves (ca. 25 mg) were heated in boiling 'Tris' buffer for 15 sec, chilled in ice, homogenized in the cold, shaken with CC14,, and centrifuged. ATP was estimated on the clear supernatant, using controls also containing CC14 (33). Weiner (73) employed special procedures to minimize the rapid breakdown of labile phosphate compounds vhich occurs in the central nervous system of rats killed by decapitation. The animals were frozen rapidly by imrsion in liquid oxygen, the brain was chipped out and extracted in cold perchloric acid, and the acid was precipitated with KOH. The change in optical density at 251 and 265 mu upon addition of adenylic acid deaminase was assumed to be equivalent to AMP in the sample. Nykinase was then added, and the change in OD265 MS proportional to one half the amount of AEW in the sample. Blanks and known mixtures of nucleotides were run with each series, and corrections were made for slight non-specific change in OD251' due to addition of enzymes. Cheng (22), working with trichloroacetic acid extracts and perchloric acid extracts (1 ) of nerve trunks, coupled the following enzyme systems, each followed by a heat inactivation step: (a) Pyrophosphorolysis of ATP in presence of sulfurylase (64) and pyrophosphatase (41) (b) Phosphorylation of ALP to ATP with creatine phosphokinase (42) (c) The final assay of ATP (phosphorylated AMP) with luciferase (71).

Minard and Davis (58) used even More exacting methods of extraction of rat brains than did Weiner (73) for the separation of nuscleotides, especially ATP. They immersed the rat suddenly in






7


liquid nitrogen for several minutes. The brain was chipped out, collected in a mixture of Dry-Ice and acetone, quickly pulverized, and extracted in trichloroacetic acid at 0 C. Chromatographic separation of nucleotides was based on the method of Hurlbert et al.

(38). Phosphorus measurements and OD260 readings were made on each fraction to calculate the total nucleotide concentration. There was no correction for formate, which absorbs strongly in the ultraviolet region. The positions of adenine, guanine, and uridine nucleotides were determined by comparison with chromatograms of known nucleotides. The peak fractions were pooled, subjected to hydrolysis in 1 N El, and the individual bases were identified by paper chromatography. Portions of the eluate fractions containing AMP, AWP, and AP were passed through columns of Dowex 50 resin (hydrogen form) to remove ammonium ion, evaporated to dryness in vacuo to remove formic acid, and hydrolyzed. Components of the hydrolyzates were separated with a Dowex 1-chloride column developed with 2-amino-2-methyl-1,3-propanediol and HCl. This system can separate adenine, guanine, CHP, UWP, and AMP.

Bishop et al. (19) separated the nucleotides in trichloroacetic acid extracts of whole blood, according to Hurlbert et al. (38); but non-gradient elution was used. The material in each peak was evaporated to dryness on a steam bath, and the free purines and pyrimidines liberated by hydrolysis for 1 hour at 72 C in 72% perchloric acid. Neutralized solutions were chromatographed in the isopropanol-HC1 system of Wyatt (74) followed by the butanol-NH40H system of Markham and Smith (51). The bases were eluted with dilute HCl, and their spectra determined.

Cohn (23) introduced ion exchange column chromatography into the






8


nucleotide field. Adenylic, uridylic, guanylic and cytidylic acids were eluted from Dowex 1-chloride polystyrene anion exchange resin columns by dilute HCl-NaCl solutions in a sequence roughly predictable from pK values. Chromatograms were prepared by measuring OD260 of each fraction collected and plotting these values against the volume eluted. Cohn and Carter (24) similarly eluted AMP, AIP, and ATP. Paper chromatograms were developed on material from peak tubes, using an isoanyl alcohol-disodium phosphate mixture and the nucleotides detected by ultraviolet fluorescence.

Horecker and SmYrniotis (36) reported a partial separation

of R5P and a similar substance later found to be ribulose-5-phosphate on a Domex 1-formate column by elution with 0.1 M formate buffer. This was a pioneer experiment in the elucidation of the pentose phosphate shunt. The pentose phosphates were produced when a purified yeast enzyme was mixed with 6-phosphogluconate.

Benson et al. (16) separated F6P from FDP on a Dowex 1-chloride column eluted with 0.1 1_ NaCl. Kbym and Cohn (39) reported the separation of synthetic mixtures of sugar monophosphates (GlP, G6P, F6P,R5P) by forming borate complexes simultaneously with elution from Dovex 1-chloride column with four chloride solutions. Hexose phosphates were estimated by the anthrone method (59), R5P by the orcinol method (21), and phosphorus was assayed according to Fiske and SubbaRow (30).

Goodman, Benson and Calvin (32) used the above techniques of Khym and Cohn (39), Benson et al. (16), and Horecker and SmWrniotis

(36) to study photosynthetic products in ethanol extracts of the alga, Scenedesmus. A synthetic test mixture of F6P, FDP, and 3PGA, and a






9


mixture of 3PGA, FEP, and Ribulose-1, 5-diphosphate was separated on Dowex 1-chloride by elution with a dilute NaCl-HCl solution. They also separated FlP, F6P, and G6P by elution with 0.1 M Na2B407 from a column converted to the borate form by eluting a Dowex 1-chloride column with Na2B407. Finally, they chromatographed a simple synthetic mixture of the above sugar phosphates mixed with an extract from algae grown in the presence of P32. Radioactivity was found in peaks corresponding to various esters, indicating the natural incorporation of phosphorus in those compounds by the algae.

Aisenberg ( 2 ) used Dowex 1-chloride columns mainly to remove free sugars from the glycolytic intermediates in the acid-soluble fraction of high-speed supernatant of brain extract. The free sugars were washed through the column with water, the esters were eluted batchwise with 0.1 N HCl, the eluate was concentrated by lyophilization, and G6P, F6P, G3P and hexose diphosphate were enzymatically assayed (25)Bergkvist and Deutsch (18) separated synthetic mixtures of mono-, di- and triphosphates of adenine, guanine, and uridine by applying seven successively increasing concentrations of format buffer. OD260 of each fraction was plotted against volume through the column and identification of each peak was made by comparing its UV spectrum with spectra of known nucleotides.

Diedrich and Anderson (28) used the method of Goodman et al.

(32) to separate galactose-l-phosphate from other common hexose monophosphates in a synthetic mixture. They described two methods to remove the troublesome borate ion from column eluates. They found, as had Helbert and Brown (35), that the color in the enthrone assay (65)





10


varied from hexose to hexuae, a they computed values from each parent sugar. The method they generally used to monitor fractions was based on Dische et al. (29). This modification enabled them to distinguish pentose from hexose in mixtures.

Diedrich and Anderson (27) used similar methods in studying the appearance of galactose. Trichloroacetic acid extracts were resolved into several anthrone-positive peaks, each of which was revealed by paper chromatography to contain several unidentified compounds. The fractions of the first two peaks were pooled, and tested enzymatically for galactose-l-phosphate.

Tiselius et al. (9 ) discussed some limitations of stepwise

elution and introduced a new general procedure for *gradient elution " of mixtures of carbohydrates. Gradient elution has since been successfully applied to mixtures of organic acids, amino acids, peptides, proteins, and nucleotides. Iakshmznan and Lieberman (43) stated the advantages of using concave gradient elution, in which the rate of change of eluent concentration increases with the concentration. The more easily eluted substances are spread apart in the chromatogram, and, since the concentration toward the end of the chromatogram increases rapidly, the more tightly bound substances are eluted without undue delay. This system also produces narrow peaks with a minimum of tailing. Pontis and Blumson (62) used concave gradient elution to separate a synthetic mixture of nucleotides on an ion exchange resin. Bock and Nan-Sing Ling (20) described systems generating several types of elution gradients.

Hurlbert et al. (38) described a system of "extended gradient elution." The reservoir, containing the concentrated eluent, and






11


the miring vessel, initially containing wter, wre designed so that the contents of the reservoir could be changed at intervals. Several ammium formte buffers vere used to separate the nucleotides in synthetic mixtures. The nucleotides in a perchloric acid extract of rat liver wre similarly separated. Nkrtonosi (55) used extended gradient elution (38), a Dowx 1-column (bicarbonate form), and K03 eluent solutions to separate a synthetic mixture containing AN, AIW, ATP, NE, IP, TPN, PP, 1I, and ITP. Bicarbonate as removed from eluate fractions by neutralizing with perchloric acid, followed by centrifuition of the potassium perchlorate precipitate. Adenine and madnine nucleotides wre assayed from OD values; iWn, IP, TPN, PP, IP, and MP wre assayed by IP and TP determination.

' Hills (5T) used extended gradient formate chromatograpy to

separate several nucleotides in red blood cells. Monitoring as done by measuring OD0 of each fraction. Numerous chromatograms of known nucleotides vare run for comparison. Several nucleotides wre quantitatively estimated by using the appropriate extinction coefficients after they vare first identified by comparing their absorbency ratios in the region from 255 to 290 m with the ratios of knomw nucleotides and derivatives. (61)

Wad9 (72) devised a complicated system, employing pumps which

operated automatically in response to a pS-sensing device, to separate various synthetic mixtures of pbosphate esters. The system ma desiped to che the AE over a much wider ranG. than could be obtained by changing buffer concentration.

Bartlett (12) reported a very extensive investigation of glycolytic intermediates and co-enzymes in red blood cells. This work






12


vs based on anion exchange chromatography, but many related techniques were employed.

Studies on phosphorylated compounds in plants to the year 1952 are reviewed by Albaum ( 4). In the earliest studies, the acidsoluble fraction was assayed for inorganic phosphorus and organic phosphorus. ATP was measured by determining the increase in IP after hydrolyzing the acid extracts for 7 minutes at 100 C in 1 N Hl; it was assumed that all phosphorus labile under these conditions is ATP phosphorus.

In 1943 LePage and Umbreit ( 40 presented a system to determine specific P-compounds in synthetic mixtures of pure compounds. This method as based on the finding that IP, ATP, AP, PGA, and FDP form soluble barium salts under the sae conditions. They were able to apply this method to trichloroacetic acid extracts of Thiobacillus thioxidans by carefully controlling the quantity of tissue and reagents. IP was determined directly. PGA was estimated by a colorimetric reaction then thought to be specific. FM and F6P were estimated by a colorimetric method. GlP was estimated as "seven minute phosphorus." IWN was estimated from the concentration of nicotinamide. AMP was estimated from nitrogen and ribose determinations, after correcting for WNq. ATP and AIW ere measured by determining total nitrogen as an index of purine present, peantose assay, and ratio of labile phosphorus to total organic phosphorus, after deducting the phosphorus resulting from other compounds present. Correction had to be made for phosphorus bydrolyzed from FW (about 27%). The remaining phosphorus vas assumed to be G6P. Despite the large number of assumptions and corrections made by TL Page and






13


Umbreit, they reportedly accounted for over 90% of the acid-soluble phosphorus.

Since this general method worked in bacteria (and anime- tissues), Albaum and Umbreit ( 8) attempted to apply it to higher plants (oat seedlings). Large amounts of non-specific absorption resulting from the presence of various polysaccharides, frustrated theri efforts to apply colorimetric sugar assays. Also, after applying all the corrections, much phosphorus still could not be accounted for. It wa known that cereals contain quantities of phytin but correction for phytin still left some phosphorus unaccounted for.

Albaum and Ogur ( 5) tried to isolate ATP from oats by traditional methods of fractionation with barium and mercury. Although polysaccharide could be removed from animl tissues by precipitation with ethanol, this was not true of the oat extracts; polysaccharides were carried along in each precipitate and each supernatant. Also, barium phytate was precipitated under almost the same conditions as ATP. Finally, there was much unknown material absorbing nonspecifically in those parts of the ultraviolet spectrum where the absorption of nucleotides was maximum. Albaum et al. solved the problem neatly by turning to a non-cereal source of plant material, the mung bean. This material yielded ATP of about 70% purity. The Albaum group (6) also tried to apply the fractionation to Euglena. The presence of inorganic phosphate, pyrophosphate, metaphosphate, AMP, ADP, ATP, DPN, GlP, F6P, PGA, and riboflavin phosphate was reported. Marre et al.(53) studied the effects of auxin treatment and pollination on levels of ATP and some glycolytic intermediates in tomato ovaries, by the methods of LePage and Umbreit (46) .









Most of the early fractionation procedures required large amounts of plant material. In the isolation of ATP from mung beans 5 to 10

pounds were used. Later column chromatography and paper chromatography were used with smaller samples of material. Albaum(4), working with mung bean seedlings, prepared trichloroacetic acid extracts from

3 g of tissue, precipitated the P compounds with ethanol and barium, and precipitated barium with H2SO4. The solution was poured through a small column of Dowex 1-chloride, and eluted stepwise with five increasingly concentrated chloride solutions, displacing adenosine, adenine, AMP, AWP, and ATP in succession.

Laughman and Martin (44) separated acid-soluble organic phosphorus compounds from roots after short periods of absorption of P32 by young barley plants. Extraction by 0.2 N HCl, 0.5 N trichloroacetic acid, or 80% ethanol gave similar results. Separation was performed by paper chromatography by the method of Hanes and Isherwood (34). F-ive radioactive spots were resolved by a butanol-water-picric acid system. Each had the appearance of a single compound, but each was composed of two or more substances, which could be separated by the use of other solvent systems. Unknown radioactive compounds were mixed with the pure unlabelled forms of suspected compounds. Chromatograms were then run in solvent systems separating the known substance from others. Active areas were compared with those developed by a molybdate spray,

revealed under UV light.

Several investigations have dealt with the assay of phosphate

esters in the fruit and seedling of pea, Pisum sativum L. Rowan et al.

(68) reported changes in levels of ADP, ATP, HMP, and Hfl in 4_ek,1oric acid extracts of pea fruits at various stages of maturation, by






15


Slater's procedure (70). There as a possibility of interference in the determination of ATP by UTP and aIP (67), but, since paper chromatograms of the nucleotides shoved the latter materials to be present in small amounts, no correction was made. Rowan (66) extended the investigation to the quantitative determination of uridine mono-, di-, and triphosphates, as well as ADP and ATP. In this work a Iarge amount of material was used (250 g to 2 kg).

Marre and Forti (52) extracted stem sections of seven-day-old pea seedlings in perchloric acid. The extract was treated according to the method of Crane and Lipmann (26) with "Norite A" charcoal, which absorbs nucleotides but not sugar phosphates. The nucleotide-containing Norite A was filtered, heated in a boiling water bath for 10 minutes in 1 N Wl, and IP was assayed. Crane and Liiuann (26) extended this procedure to measure ADP and ATP labile phosphorus, but other nucleotides might be present in such an extract, which would result in high readings.















MATERIALS AND M)DS


Plant Materials

Dried kernels of hybrid seed corn (Zea mas L., var. Funk's

G-50 and G-740) were rinsed thoroughly, and soaked with aeration in tap water for 24 hr. The kernels were rinsed again and then placed individually on trays lined with thoroughly wetted filter paper. Each tray was covered and the kernels were allowed to germinate in the dark at 25 C for 72 hr. The distal 2 cm was cut from the primary root of each seedling and collected in ice-cold distilled water. The root tips were filtered in a Buchner funnel, washed once with ice-cold water, gently spread on filter paper to remove adhering water, and weighed to the nearest 0.1 g. A typical harvest of 650 to 700 root tips yielded about 20 g of tissue. Preparation of Extracts

The weighed root tips were thoroughly ground for about 3 min in 10% trichloroacetic acid (1 ml/g of tissue) in an ice-cold mortar. The resulting slurry was centrifuged, and the supernatant liquid was decanted. The residue was suspended in 5% trichloroecetic acid (1 ml/g) and centrifuged. The combined supernatant fractions were retained; the residue was discarded. All manipulations to this point were performed at 0-5 C. The trichloroacetic acid was removed by four extractions with two volums of cold ether; the pH of the extract as then about 3.5. A thin stream of nitrogen was bubbled through the



16






17


extract until the ethereal odor as not discernible, and 5 N EH40H as added to pH 6.8-7.0 This extract us passed through a Dovex 50 coluin, and followed by 50 ml of mter; the combined filtrate as neutralized to pH 6.8-7.0. One to four extracts were used in chromatography. The extracts could be stored several weeks at -20 C. Preparation of Ion Exchange Colums

Trimethylamonium polystyrene ("Dovex") resins (Dow Chemical Co.) were obtained in a purified form (Bio-Rad Laboratories, Richmond, Calif.). Two types of anion exchange resins and one type of cation exchange resin were used.

One anion exchange resin, AG 1-X8, 50-100 mesh, was suspended

in meter and the extremely fine particles were decanted. Fourteen-cm resin beds were formed in 1 X 30-cm glass chromatographic tubes with sintered glass retainers (Emil Greiner Co.). Uniformity of the many column prepared during these investigations was approached by stirring thoroughly the thin resin slurry immediately before pouring the column, and repeatedly inverting the column while adjusting the resin bed to the desired length. The resin colums were each capped by a 9ml1 plug of washed glass wool, and eluted successively with two bed volumes of 88% formic acid (38), three volumes of wter, three volumes of 1 N NH4C1, and, finally, with water until the filtrate had the same pH (5.5-6.0) as the distilled water used, and as also chloride-free. Chromatographic columns prepared in this manner are subsequently referred to as "Dowex 1-chloride" columns.

The other anion exchange coluums, containing AG 1-X8, 200-4w0 mesh, were prepared as follows. The extreme fines were decanted and the amorphous particles were removed with a medicine dropper. One X


-1 -1,- A, - _ -





18


14-cm resin beds were poured as described above and eluted with three bed volumes of 88% formic acid, four volumes of 5 N ammonium formte (until the filtrate was chloride-free), and with an excess of water. Such resin columns are referred to herein as "Dowex 1-formate" colums.

The cation exchange column were prepared from AG 50W-X8, 100-200 mesh. Three-cm beds were poured in 1 X 30-cm tubes. The resin was eluted with 30 Ml of 2 V Wl, with water until the filtrate was chloride-free and with an excess of water. Colum Chromtography

Solutions containing anions to be separated by ion exchange colum chromatography were passed through Dovex 1-formate columns at flow rates of about 3.0 ml/min . Solvent flow was maintained by two to four pounds of air pressure from a low pressure regulator (Matheson Co.) and adjusted by a teflon-glass needle valve (Emil Greiner Co.). Connections were made with silicone rubber tubing Type R (Ronsil Co., Little Falls, N.J.) after it was found that "tygon" tubing introduced significant contamination in colorimetric analyses of the eluate fractions. The fractions were collected by an automatic fraction collector (Research Specialties Co.), with a volumetric siphon; graduated test tubes were used until the reliability of a siphon was established. Twenty-ml fractions were collected from Dowex 1-chloride columns and 10-ml fractions were collected from Dovex 1-formate columns. Each fraction was assayed for TP, and chromatogramw were always prepared to reveal the elution positions of P-containing compounds.

The anions that were adsorbed on Dowex 1-chloride columns were eluted at about 2.8 mi/min by 200-400 ml of each of the following






19


s )lations (13): 1. 1 11HC1, 0.'2 11 HC1; 0.1 1; III Cl; I. i '1,1 4 Cl,

0.5 W NHCl.

In preliminary experiments, the fractions obtained with each of the five chloride eluent solutions from a Dowex 1-chloride column, containing compounds of similar resin-binding capacity, were pooled, passed through a Dowex 1-formate column, and the anions were loited by formic acid or ammonium formate buffers. In later exeriments the Dowex 1-chloride column eluate was collected in five batches corresponding to the five eluent solutions. Each batch was neutralized with NH40H to pH 6.8-7.0, and was stored at -20 C until chromatDgraphed with a Dowex 1-formate column.

Dowex 1-formate columns were also used to chromatograph directly whole trichloroacetic acid root tip extracts without prior separation on D~wex 1-chloride columns. The fractions in a TP peak were often pooled, passed through Dowex 50 (hydrogen form) to remove ammonium ion, extracted several times with ether to remove formic acid, neutralized, and retained for analysis.

Dowex 1-formate columns were eluted at about 1.0 ml/min with

linear )r concave gradients of formic acid or ammonium formate buffer. Concentration gradients were obtained with systems diagrammed by Bock and Ling (20). A linear gradient was obtained when the crosssectional areas of the two eluent-containing vessels were the same. Concave gradients were obtained when the area (A1) of the mixing vessel was greater than that of the reservoir (A2). The ratio, A2 = C.6, was selected for use in these experiments (62). However, Al
linear gradients were used more often than concave gradients after preliminary experiments with the latter.






20


Various synthetic test mixtures of known phosphate compounds

were also chromatographed by the same procedure with Dovex 1-chloride or Dowex 1-formate systems for comparison with chromatograms of plant extracts.

Analytical Methods

Phosphorus (TP, IP, 7-min P) assay. Phosphorus was assayed

colorimetrically by measuring the blue phosphomolybdate complex. The following modification of the Gomori (31) method permitted reliable and relatively convenient assay of large numbers of samples.

For TP assay, the sample was wet-ashed by heating for 3 hr in an oven at 170-175 C in a 10 X 180-mm test tube calibrated at 10 ml and containing 1.0 ml of 5 N H2SO4 and a glass bead. Two drops of 30% hydrogen peroxide were added, and heating was continued for an additional 2 hr. Three drops of 5% urea were then added and heating continued at 100 C for 3 hr. One ml of water was add d and heating continued at 120-130 C for 1 hr. After the tube had cooled, two ml of 6.25% Na2MOO4-2H20 in 5 R H2Si4 were added, followed by 1.0 ml of 1% Elon (31) (Eastman Co.) in 3% NaSO The color formed was read, after 10 to 60 min development, in a photoelectric colorimeter (Klett Co.) using filter #64. Calibration curves were prepared using an inorganic phosphate standard solution.

IP was assayed similarly, but without heating. The temperature was kept below 22 C, and the sample was diluted before addition 9; acid, to minimize hydrolysis of labile phosphorus compounds (27).

Acid-labile, or "7-min" phosphorus was estimated by the increase in IP after 7 min at 100 C in 1 N HCl.

Assays for sugar nhosphates. Hexose was assayed by a modification


1 1- .4 ,


I





21


of the enthrone method (10). Two-ml samples were layered over 4.0 ml of 0.2p anthrone in 955 H 2S04 while chilled in an ice bath. The mixture was shaken vigorously, heated for 15 min at 100 C, and cooled in tap vater; absorbancy was read in a colorimeter (Klett Ci.; filter 60) against a standard calibration curve prepared from glucose or fructose. The mixture was usually shaken once during the heating step to remove bubbles.

Fructose was assayed as follows (10). T-, 2.0 ml of the sample

were added 0.2 ml of 1-%5 cysteine, 4.0 ml of 95 H2304, with cooling in an ice bath, and 0.2 ml of 0.1 carbazole in absolute alcohol. The mixture was shaken, heated at 60 C for 30 min, and cooled in tap water; absorbancy was read in a colorimeter (Klett C-.; filter 56), or in a Beckman IU spectrophotometer at 470, 560, 650, and 750 mu

(10) against a standard calibration curve prepared from fructose.

Pentose was determined by the following modification of the orcinol method (10). T- 3.0 ml of the sample were added 3.0 ml of

0.1 FeCl3 in concentrated HCl (vith cooling in an ice bath), followed by 3 drops of orcinol reagent (500 mg/ml of absolute alcohol). The mixture was shaken and heated for 10 to 20 min at 100 C. Absorbance was read in a photoelectric colorimeter (Klett Co.; filter 64) against standard curves prepared from arabinose, ribose, or R5P.

Assay for glycerates. Glycerates were analyzed as follows (14). The sample, in a volume of 0.2 ml, was added to 5.8 ml of 0.01% 4,5dihydroxy-2 ,7-naphthalene-disulfonic acid (' hromotropic acid). The mixture was shaken and then heated for 30 min at 100 C, cooled, and the absorbency read in a colorimeter (Klett Co.; filter 69) or Beckman






22


DU spectrophotometer at 690 mu against a known standard solution.

Enzymatic assays for phosphorus compounds. An enzyme, or a mixture of enzymes, was used to assay G6P, F6P, and UOPG. In each case, the enzyme activity vas tested against known substrate preparations. The change in optical density vas measured by a Beckman model W spectrophotometer using a quartz cuvette having a 1-cm light path. The level of substrate in a sample was computed from the molar absorbancy value of the reduced pyridine nucleotides at 340 mu (6.22 X 103), and this value was multiplied by the appropriate factor to give the results reported herein. A method for the determination of millimicromolar amounts of AP is described in more detail.

G6P and F6P. G6P vas assayed by following the reduction of

TPN in the presence of glucose-6-phosphate dehydrogenase (G6PD) ( 37).

G6PD
G6P+TP > 6-phosphogluconate+TFIe+H+


The amount of G6P was calculated from the increase in OD34 according to the expression

( OD)(3.0)
G6P (umoles) =6.22


A typical reaction mixture contained 1.0 ml of 0.04 _ glycyl glycine buffer (pH 7.5), 1.0 ml of 0.02 M MgCl2, 0.2 ml of TPN (4 mg/ml),

5 ul of G6PD (Sigm Type V), and G6P, in a total volume of 3.0 ml.

P6P was assayed in the same wq in the presence of G6PD and phosphohexose isomerase (50).

PHI
P6P -> 06P


Uridine diphosphate glucose (UDPG). UDPG was measured by






23


following the reduction of DPN in the presence of uridine diphosphate glucose dehydrogenase (UWPGD), which is specific for the reaction

UDlPGD
UWPG+2MP ) U&E lucuronate+2MPBE


UWGD was prepared from calf liver, according to Maxwell et aal. (56) The amount of UWPG was calculated from the increase in OD34o according to the expression

( OD)(3.0)
UVPG (umoles) = 12.0


A typical reaction mixture contained 0.06 ml of DPN (35 mg/ml, 0.3 ml of 1 M glycine buffer, pH 8.7, 0.5 ml of UGD, the sample containing UIWG, and water to a final vol of 3.0 ml.

Microdetermination of ATP with firefly lantern extract. The intensity of the luminescence is proportional to the ATP concentration

(48). As little as 2 X 10 umoles of ATP can be measured with a Farrand photofluorometer, using an extract prepared from firefly lanterns.

The extracts were prepared by the following modification of the method of McElroy (R ). Fifty mg of dehydrated firefly lanterns (Sigma Chemical Co.) were ground in a small glass homogenizer (Kontes Glass Co.) for 3 min in 1-1.5 ml of 0.1 M Na2AsO4 buffer, pH T.4, at 1-4 C. The extract was transferred to a graduated test tube. The homogenizer was rinsed twice with cold buffer, the rinsings and extract were combined, and the volume was made up to 5.0 ml with buffer. Cell debris was sedimented by centrifugation and discarded. Fifty mg of MgSO4. 7H O were dissolved in the supernatant liquid. This









preparation is herein referred to as firefly lantern extract (FFE). All manipulations were done in the cold. Filtration of the homogenate (71) was not as convenient as centrifugation, and activity of the filtered preparation was about 50% lower. The FFE retained 90% of full activity for 1 week at 5 C. A typical reaction mixture contained 0.2 ml of FFE, the sample to be assayed, and water in a total volume of 0.8 ml. Each preparation was calibrated against known ATP samples. The galvanometer was read 30 sec after adding the sample. Full scale deflection (10.0 units) was usually produced by 0.2 mumoles of ATP.

The presence of transphosphorylases in the FFE was confirmed by several experiments in which GTP or GTP plus AWP were added instead of ATP (1).

Trichloroacetic acid extracts of corn root tips were assayed in the following manner. A portion of the extract was diluted with water to a suitable concentration, and a 0.2 ml sample was assayed as decribed above. The galvanometer reading was multiplied by an appropriate factor to determine the total ATP in a known weight of plant tissue.

Single root tips were assayed as follows. A weighed, 2.0 cm

root tip (25-30 mg) was homogenized in 1.0-1.5 ml of 1.5 M perchloric acid for 30 sec at l-4 C. The homogenate was transferred to a small centrifuge tube, and the homogenizer was rinsed three times with about 0.5 mil of cold water. The pH was adjusted to 7.2 with KOH (1.5 M and 0.15 M), and the extract was centrifuged. The supernatant fluid was transferred to a graduated test tube; the precipitate was suspended in cold water and centrifuged. The supernatant fractions






25


were combined, and made up to 5.0 Ml with ater. All manipulations were done in the cold. A 0.1 to 2-ml portion of this extract vas assayed with FFE .

Analysis from UV absorbency measurements. Absorbency was measured at 260 and 290 mu on the fractions eluted from Dovex 1 colums. Whereever the ratio 260/290 was high, the presence of a nucleotide was suspected, and measurements at additional wavelengths were taken to characterize the nucleotide (61).
















RESUIMS


Dowex 1-Chloride ChronatograpIa of Knomw Compounds

Test solutions containing mixtures of known P compounds were

chromatographed with Dowex 1-chloride columns. The elution sequences were similar to those previously reported (13): AMP, G6P, F6P, and IP were eluted by 0.01 1 HC1; ADP was eluted by 0.02 K HC1; FDP was elated by 0.1 N NHjCl; ATP was elated by 0.5 N NH4Cl. The positions of the compounds were determined from chromatograms drawn from TP assay and OD260 measurements on each fraction. Another Dowex 1-chloride coluam was elated with the five solvent systems. Each fraction of the "blank run" gave a zero response to TP assay and had negligible absorbance at 260 mu.

The enthrone and cysteine-carbazole assay methods were applied to the P-containing fractions. Some of the fractions showed surprisingly high absorbency readings, due to the fortion of bubbles. This result was most severe in the fractions containing the highest concentrations of ammonium ion, but could be minimized by thorough shaking of the test tubes with a mechanical test tube shaker several minutes before absorbancy measurements were made.

Dovex l-Forite Chr9Mtograpiy of Knovi Compounds

A "blank run," using 0 to 1 N formic acid, showed each fraction to be negative for TP. Absorbency increased greatly with formate concentration in the 200-240 mu range, but was negligible in the near


26






27


UV range. Anthrone and cysteine-carbazole assays on the same fractions gave low but erratic readings due to bubble formation in the presence of formate ion; vigorous shaking was necessary to minimize errors from this source.

A test solution containing AMP, GlP, G6P, F6P, R5P, and IP was

chromatographed. The anthrone, cysteine-carbazole, and orcinol assay methods were applied to each fraction; TP was also assayed on each fraction. AMP was measured from 0%60 readings. The compounds were eluted in the above sequence; AMP and IP were eluted separately, but the sugar phosphates overlapped.

When using 10 min heating time with the orcinol method, and

reading immediately, GlP, G6P, and F6P all gave virtually unmeasurable readings (less than l0 of the readings given by R5P). Using a 20 min heating time increased the sensitivity by 3%, but increased interference from hexose phosphates by 10%. The color was stable; readings after 24 hr at room temperature indicated intermediate sensitivity and specificity. The assay appears to be extremely specific for pentose in the presence of large amounts of hexose. Cysteine-carbazole method was not specific for fructose phosphates, as reported by Bartlett

(13), but absorbency readings for GIP and G6P were about 35% as high as for an equivalent amount of F6P. Only a trace of reaction with cysteine-carbazole was given by R5P.

Ar other test solution, containing UUPG, FDP, 2PGA, and 3PGA, was chromatographed with a Dowex 1-formate column, which was eluted with 0 to 4 N formate buffer, containing four parts of formic acid and one part of amonium formate (pH 3.0). UWG and the two glycerates were eluted simultaneously.






28


Dowex 1-Chloride Chrmatography of Corn Root Tip Extracts

Table 1 shows the levels of TP and IP in pooled trichloroacetic acid extract of 63.2 g of root tips, before and after passage through a )owex 50 column; recovery of IP was nearly quantitative, while 15% of the TP was retained on the cation exchange resin. Table 2 shows TP and IP levels in several Dovex 50-treated root extracts.

Figs. 1-4 show the results of Dowex 1-chloride chromatography of root tip extracts. The composition of the eluates could not be determined from these data. It appeared from these results that (a) only a crude separation was obtained by the Dowex 1-chloride colum

(b) the compounds of interest were masked by the presence of various materials giving color formation with TP and sugar phosphate analyses, and others having absorption in the UV spectrum (,:) formation of bubbles obscured results in the three sugar assays. Dowex 1-Formate Chr;matograhy Df Eluates from Dowex 1-chloride Soparatins o: Corn >ot Tip tractss

Since Dowex 1-chloride chromatography apparently did not separate sufficiently the phosphorus compounds of interest, and since considerable aqnounts of unknown materials appeared to mask the results, the solutions eluted from Dowex 1-chloride columns were chromatographed again on D)wex 1-formate columns: each of the five pooled eluates from the chloride colums was passed through a Dowex 1-formate column, and the anions were elated either with formic acid or a formic acidammonium formate baffer.

0.01 IT HC1 section. Fig. 5 shows the results obtained from the rechromatography of the 0.01 N HC eluate from a trichloroacetic acid extract of 18.6 g of root tips, using 0 to 1 N formic acid for elution. Similar results were Dbtained from extracts of 19 to 71 g of root tips.


wkifimwwl






29


TABLE 1


PHOSPHORUS COI'ENT OF A TRICHIDROACETIC ACID EXTRACT OF 63.2 GRAMS
OF CORK RIKT TIPS, BEFORE AND AFTER TREATMENT WITH DOWEX 50



P, umnoles/g 1% Recovery
IP TP IP TP


Before Dovex 50 3.09 6.64 -- -After Dowex 50 3.03 5.63 98 85


A





30


TABLE 2

PHDSPHDRUS COUTT OF SEVERAL TRICHWROACETIC ACID EMRACTS OF CORK
ROOT TIPS, AFTER TREATMENT WITH DOWEX 50



Wt of root, g IP, uinoles/g TP, umoles/g

57.1 4.C4 7.50
74.4 3.03 5.24
63.2 3.03 5.63
29.6 2.88 7.10

Av. 3.24 Av. 6.37


I








31


.5




.4




.3


0

.2




.1





.1 .2 '.4 .4 .d .6 .7.A . 1.0 1.1 1.2 1.3
.01 N MCI .02 .JN .2N .SN
NHCI NH4CI NH4CI NH4CI
LITERS THROUGH COLUMN


Fig. 1. TP


0


.15






.10


.05t


-


A .4 -s .4 .1 .6 .1 .A.4 1.0 1. 1.2 1.3
.01 .02 .JN .2N .5N
NHCI NHCI NH4CI NH4CI NH4CI LITERS THROUGH COLUMN



Fig. 3. Anthrone

(hexose)


1.75


1.50


1.25
0
A

0


.25[


A1 t~ '. A .3 .6 .7 1.6 .0 1o0i.112U143 .01NHCI .02N IN .2N SN
HCI NH4CI NH4CI NH4CI

LITERS THROUGH COLUMN


Fig. 2. OD260


.06 .04






.02


.1 .1 .3 .4 .5 .6 .7 .8 .9 1.0 1112 13
.01N HCI.02NHCI .1N .2N .SN
NN4CI NH4CI NH4CI
LITERS THROUGH COLUMN




Fig. 4. Cysteine-carbazole

(ketose)


Figs. 1-4. DOWEX 1-CHILRIE CHOMATOGRAPHY OF TRICIDROACETIC

ACID XTRACTS OF COIN ROOT TIPS. The Dowex 50-treated extracts were run through Dovex 1-chloride columns, and the anions were desorbed by elution with the indicated solutions.


.75


A









I


1






32


.35s
2

.30







.20



0 .1s.05
.1. .20.2










LITERS THROUGH COLUMN





Figuare 5. DOWE 1-FORMATE CE30MATOGRAPHY OF THE WIFAK ACID PM)SPHATE COMPTOU= FROMl A TRICHWROACETIC ACID EXTRACT OF 18.6 GRAM OF CORNT ROOT TIPS. The pooled, neutralized, and diluted 0.01 N HC1 section of a Dovex 1--chloride separation was run through a Dowex 1-formate column and the adsorbed anions were elated with 1-3 1 of concave gradient 0 to 1 11 formic acid. Assay methods: TP: Anthrone: --,Orcinol.






33


The chromatogram was plotted from TP, anthrone, cysteine-carbazole, and orcinol values. The color formed with the enthrone and cysteinecarbazole reagents was compared with the color formed iith fructose standard solutions, and was masured with the Klett colorimter. The TP was recovered in two well-defined peaks. Table 3 shows the recovery of TP from four root extracts. Linear and concave gradient elutions proved to be equally satisfactory; the only apparent difference was an overall shift of the position of the elution peaks.

A large amount of absorbance in the enthrone method appeared in the first 400-500 ml of eluate. This was probably due to the presence of non-phosphorylated carbohydrates, since no phosphorus was detected in these fractions. The cysteine-carbazole and enthrone assay methods gave similar results when applied to the fractions of Peak 1; addition of the values obtained with those fractions gave 1.46 umoles of ketohexose/g of roots with anthrone and 1.45 umoles of hexose/g of roots with cysteine-carbazole. The fractions in Peak 2 were essentially negative with enthrone, except for a loy "background" absorbance probably due to the persistent elution of non-phosphorylated carbohydrates.

The orcinol method of pentose assay, measured against a R5P stock solution, gave a total of 0.387 umoles of pentose/g of roots, with the peak values at the same elution position as Peak 1. The orcinol result was about what would be expected from the amount of hexose monophosphate present, as calculated from anthrone or cysteine-carbazole methods. There is, then, no evidence for the presence of measurable amounts of R5P.

Assay for IP was negative for the fractions in Peak 1; IP assay

on the fractions in Peak 2 gave the same values as TP assays (Table 3),

indicating that Peak 2 consists of IP.










TABL 3


TP RECOVERY IN THE DOWEX 1-FORMATE CHROMATOGRAPHY OF THE 0.01 N HC1 SECTION FROM DOWEX 1-CHIORIDE
CHROMATOGRAPHY OF FOUR CORN ROOT TIP EXTRACTS. (See Fig. 5)



Peak 1 Peak 2 Total
Weight of roots, g TP, umoles/g TP, umoles/g TP, umoles/g TP Recovery

63.2 0.83 2.72 3.55 94%

22.7 0.89 3.86 4.75 --18.6 1.08 3.24 4.32 89%

71.1 1.04 2.65 3.69 --Av. 0.96 3.12 4.08


U)






35


Only the first 200-300 ml of eluate absorbed very significantly at 260 mu; no phosphorus was detected in those fractions.

The Peak 1 fractions from an extract of 22.7 g of root tips were tested for ketose by the cysteine-carbazole method using the Beckman IDJ spectrophotometer, with the following results. Readings were taken on each fraction at 4.70, 560, 650, and 750 mu (10). Optical density readings were somewhat lower at 650 than at 470 mu, indicating no triose phosphate was present in the fractions tested. There was negligible absorption at 750 mu. Based on the value 0.6 for 0.1 umole of ketohexose (1-cm light path), the ketohexose in Peak 1 was computed to be 0.39 umoles/g. This accounted for 43% of the TP in the Peak 1 fractions. The procedure was repeated with the fractions from another root extract (71.1 g); 40% of the TP was accounted for as ketohexose based on cysteine-carbazole assay. Since the cysteine-carbazole method was found in preliminary experiments to suffer from rather high levels (35',) of interference from GlP and G6P, the absolute values obtained in a mixture did not seem significant in themselves. However, inspection of the ratios of cysteine-carbazole readings/TP content computed for each individual fraction showed the highest values in those fractions corresponding to the descending slope of Peak 1. This is the elution position where F6P has been reported to occur (13), and which was confirmed in these studies.

The pooled fractions of Peak 1 were analyzed enzymatically for GCP; the results indicated the presence of 0.58 umoles of G6P/g of root tips, accounting for 56% of the TP in the peak.

An extract of 38.3 g of roots was tested for GP by measuring acid-labile (7-min) P in the fractions in Peak 1 (Fig. 6). All


.1I -


PEW







36


.1 3~ 1 .4 .5 .6 .7 . 9 1.11 12 LITERS THROUGH COLUMN


Figure 6. DOWEX 1-FORMATE PLATES (FMA 1) FROM AN ERACT (See Fig. 5 ). Linear gradient. acid-labile (7 min) P.


CHROMATOGRAPHY OF THE SUGAR M)NOPHOS OF 38.3 GRA1S OF CORK ROOT TIPS. Assay methods: -, TP;


.6F-


-4


19
mi


I


0


.3F-


.1


- I


.sF


.2






37


fractions were negative for IP; i.e., before hydrolysis. Determination of 7--min P on the same fractions gave peak values in the region of the ascending slope of Peak 1. This is the elution position previously reported for GlP (13), and confirmed in these studies. No other phosphorus c compound is known to be elated in this region, and none of the other sugar phosphates is appreciably labile after 7 min in 1 N HC1l at 100 C. Total GIP was computed to be 0.05 umoles/g of root tips.

0.02 N HC1 section. Fig. 7 shows the results obtained from the rechromatography of the 0.02 N 1KM eluate from a trichloroacetic acid extract of 57.1 g of root tips, using 0 to 1 N ammonium formate for elution. Recovery of P was 60Y, about 90% of which was in the two peaks having Tmaxinmum values at 490 and 720 ml.

OD260 and OD290 was measured on each fraction. Infinitely large readings were obtained in the first 300 ml at both wavelengths. The UV spectra of these fractions did not resemble those of the nucleotides, and very low levels of P were found in those fractions. Another tall UV peak having the sa characteristics, occurred at 770-810 ml.

Three fractions, at 480, 490, and 500 ml, (Fig. 7) were assayed for glycerate by the chromotropic acid method (14). In each case, the spectrum was the same as the characteristic spectrum produced by known MPG under the same test conditions. Further, the ratio of glycerate to TP was about 1:1 for each of the three fractions. From this evidence it was concluded that the three fractions contained essentially pure MPG; the concentration was calculated to be 0.01 umoles of MPG/g of root tips.

The UV spectra of the fractions collected at 720 to 750 ml


m~-t






1


A .6 .7* .6 . 1I .'"


LITERS THROUGH


COLUMN


Figure 7. DOWEX 1-FORMATE CHROMATOGRAPHY OF THE 0.02 N HC1 SECTION OF A TRICHLROACETIC ACID EXACT OF 57.1 GRAMS OF COIE ROOT TIPS. The pooled, neutralized, and diluted 0.02 N HCl section of a Dowex 1-chloride separation was run through a Dowex 1-fornate column, and the adsorbed anions were eluted with 1.2 1 of linear gradient 0 to
1 N ammonium formate buffer. Assay: TP.


33


I 1 1 1 I I I I I


.20F


.15-


-I





0


.10


.05


.1


_j


-






39


resembled that cf the adenosine hosphates. The absorbancy ratio 250/260 averaged J.70 fKr these fractions and the ratio 280/260 averaged 0.16. The oH of the eluates was 6.4. These values were identical to those previously reported for ADP at pH 7.0 (see Table 4). The nucleotide content of the same four fractions was calculated from the molar absorbancy value of the adenosine phosphates, 15.4 X 10-3

(61). The ratio of TP to adenine (cD2-) of the se fractions as

2:1. A Dowex 1-formate chromatogram of an authentic sample of ADP had the peak at the same elution position. From this evidence it was concluded that the compound was ADP; the ADP level was calculated to be 0.25 umcles/g of root tips.

0.1 I IagCl, 0.2 U TM Cl, and 0.5 11 IMiCl sections. These three eluates from Dowex 1-chloride chromatography were each chromatographed with a formate buffer, using the same techniques described above. In neither instance was there an appreciable recovery of P, most probablyy due to the presence of relatively high concentrations of chloride ion. Direct Dowex 1-Formate Chromatography of Corn Root Tip Extracts

Root tip extracts, each representing 50-75 g of tissue, were

run through Dowex 1-formate columns after purification with Dowex 50 resin. The adsorbed compounds were elated with 0 to 4 N amnonium formate buffer, PH 3.j, containing four parts of formic acid and one part of amonium formate. Linear and concave elution gradients were used. The chromatograms were monitored, as usual, by TP assay on each fraction. Comparison of a concave gradient chromatogram (Fig. 8) with a linear gradient chromatogram (Fig. 10) showed the same sequence of major peaks; but the concave gradient gave better separation of the weakly acidic compounds.










TA=LE 4


ABSORBANCY MATIOS AT 250, 260, AND 280 wU


Compound OD250/260 OD280/260

pH 2 7 11 2 7 11

AEP* 0.85 0.78 0.78 0.21 o.16 o.16
CDP* o.46 o.83 0.83 2.07 0.98 0.98
UDP* 0.73 0.73 0.80 0.39 0.39 0.32
UDPG* 0.76 o.76 o.82 0.38 0.39 I.34
Peak 6/7*** 0.83 0.79 0.83 0.47 o.45 o.43

*Pabst Laboratories, Circular OR-T, Ultraviolet absorption spectra of 5'-ribonucleotides, March 1955.
**Sigma, 90%.
**See Figs. 8 and 11.









I I I I I I I I I I I I i I I


.8



.7



.6



.5
L"


.4
.a .

0 -3
.3







-1-13U4 5 6 7 8 9.




.1 . .3 . . .7 . . 1.0 1.1 1.2 1.3 14 1.5 1.6 1.7 1.8 1.9 2.0

LITE RS THROUGH COLUMN Figure 8. DIRECT DOWEX 1-FRMATE CHROMATOGRAPHY OF A TRICHIMROACETIC ERACT OF 51.8 GRAMS OF CORN ROOT TIPS. The extract, after purification with Dowox 50 (hydrogen form), was passed through a Dowex
1 -formate colium, and elated with 2.1 1 of concave gradient 0 to 4 N formate buffer containing four parts of formic acid and one part of ammonium formate (GH 3.0). Assay: TP.


I I


I I I I I I I I I I I j I I I I











I 11 1 1 1 1 1


- 260


290


-A -i . .4 . .4 .7. . 1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2.0

LITERS THROUGH COLUMN


Figure 9. CORN ROOT TIPS.


DIRECT DOVJEX 1-FORMATE CHROATOGRAPHY OF A TRICHIWROACETIC ACID EXTRACT OF 51.8 GRAM OF (See Fig. 8). Aseay: OD260 and OD2901 as indicated.


0
04

0

0
0
4
cm
a
0


1.50 1.25



1.00-


.75F-


.50 .25


'260





290






43


11 1 ' ' I . I I . I I I .


1.1


1.0


.9


-I





A.


0
X


.8


.7


.6


.5


.4-


.3


.21


.1


I 34 5 6 7 8 9 10 11 12




.1 . . . . .0 1.0 1'. 1 1.2 1.3 1A 1.5 1.6
L ITERS THROUGH COLUMN


Figure 10. DIE DOI,= 1-F014MTE CHROMATOGRAPHY OF A TRICHIDROACETIC ACID EXRACT OF 71.9 GRAMS OF CORN ROOT TIPS. The procedure followed was described in Fig. 8; linear gradient elation. Assay: TP.





44


In a typical experiment (Fig. 8), using 51.8 g of root tips, 6.75 u1 les of P/g of root tips were added to the colurm, and 5.52 umoles (82%) were recovered in the individual fractions. Of the P recovered, from averages of several runs, 4.92 umoles (8%) appeared in the four largest peaks, as follows: Peak 3, 0.93 umoles/g; Peak 4, 3.61 umoles/g; Peak 7, 0.70 uraoles/g; Peak 10, 0.25 umoles/g. Peak 3 corresponded to the hexose monophosphate peak (Peak 1) in Dowex 1-formate chromatography of the 0.31 N HCl Dowex 1-chloride eluate (Table 3). Peak 4 was IP, corresponding to Peak 2 in the same table.

The fractions of Peak 3 were pooled, freed of ammonium and formate ions, and analyzed enzymatically for F6P. Analysis showed the presence of 0.15 u1les of F6P/g of root tips: this accounted for 14% of the

TP in Peak 3.

Fig. 9 shows OD260 and OD29o measurements of the eluates from concave gradient chromatography of 51.8 g of root tips. Extremely high levels of absorbency at both wavelengths were found in the first 300 ml, indicating the presence of large amounts of unknown materials absorbing in the near UV spectrum.

A UV peak, possibly representing a nucleotide, was found at 390450 ml. The position of the peak did not correspond exactly to Peak 4 (Fig. 8). Using the molar absorbency value 15.4 X 10-3, the total UV-absorbing material would be less than 0.02 umole/g, or 0.Vp of the phosphorus in Peak 4, on a monophosphate basis. The identity of the compound was not determined.

A small UV-absorbing peak was found at 950-1000 ml; it was not identified. Another small peak at 1380-1440 ml was not identified. A fairly large peak at 1600-1700 ml did not correspond to the TP





45


chromatogram, and its identity was not ascertained. Two peaks were found at 1030-1130 ml and 1750-1840 ml.

The fractions making up each peak in Dowex 1-formate direct

chromatography were pooled, ammonium and formate ions were removed, and UV absorbency was measured on each combined peak (Fig. 11). Only Peak 6/7 (combined) showed a UV absorption spectrum similar to a nucleotide. Peak 6 contained 4% of the TP in the combined peak 6/7. The ODm of Peak 6/7 was 262 mu, and the complete UV absorption spectra at pH 2, 7, and 11 (not shown) were similar to spectra of the uridine nucleotide, and unlike the spectra of the other nucleotides. The absorbancy ratios 250/260 and 280/260, given in Table 4, indicated that Peak 6/7 probably contained UIF or UDPG, or both. Enzymatic assay of the peak indicated the presence of 0.13 umoles of UWPG/g of root tips, corresponding to

0.26 moles P/g root tips; 38% of the TP in Peak 6/7 can be accounted for as UDPG.

Orcinol, anthrone, chromotropic acid, cysteine-carbazole, and acidlabile (7-min) P assay methods were applied to each of the pooled peaks. No clearcut results were obtained, because the peaks all contained impurities which interfered with colorimetric assays. ATP Assay of Corn Root Tin Extracts

The firefly lantern extract was used to measure the ATP level

in trichloroacetic acid extracts of root tips. The results indicated 0.15 umoles of ATP/g of root tips in the acid-free extract. When the

extract, containing trichloroacetic acid, was analyzed after neutralization with 0.1 M Tris buffer to pH 7.4, the apparent ATP concentration was only 20% of that observed in the acid-free extract, possibly due to a salt effect. ATP content of a perchloric acid extract of a single root tip was 0.18 umoles/g.






46


2.4
2.3
2.2
2.1


1.9




1.


1.3-5


1.2
1.1
1.0
.9 - 3.8

.7 -


.4
.3
.2
.1 -'

220 230 240 250 260 2O 260 20







Figure 11. ULTRAVIOLET SPECTRA OF TIE POOLED TP PEAKS F01M DIRECT DOWEX 1-FORMATE CHROVATOGRAPHY OF TRICHIROACETIC ACID EXTRACTS OF COIT ROOTS. The fractions of each numbered peak (see Figs. 8 and 10) were pooled, ammonium ion.was removed with Dovex 50 (hydrogen form), formic acid was removed by extracting with ether; each solution was neutralized bef -re its UV absorbency was measured.









Phowphorus Conwounds Ident4fied in the Root of the Etiolated Corm SeedlbzZ

Table 5 shows the concentrations of the P compounds found in the seedling root. The values for MPG and AMP are, undoubtedly, much too low, because about 10O% of the TP failed to adsorb to the Dovex 1-formwte column.













VI


. -- 1 41; . .





48


TABLE 5


PHDSPHATE C01UNG OF COIL 1MOT TIPS




Compound umoles/g of corn root tips


:rP* 2.65-3.86

GiP .05

G6P .58

F6P .15

UEPG .13

AEP .85

ATP .15

MPG .01


*Range of values from four extracts. Each other value from a single extract.
















DISCUSSION


Previous workers have encountered difficulties in the separation and analysis of the phosphorus compounds in plant tissues, and one of these, Albaum ( 3 ), declared the use of ion exchange chromatography for this purpose "not feasible," because of the swamping of the analyses at every stop by large quantities of phosphorus compounds of various compositions.

Considerable difficulty was also encountered in these studies for similar reasons. The corn seedling root tissue contained high

levels of phosphorus compounds of unknown composition, which vere difficult to separate from the glycolytic intermediates and nucleotides which were of primary interest. However, the results of the investigations reported in this paper indicate that anion exchange methods show prDmise for the separation and quantitative analysis of those compounds of corn seedling roots, and it is believed that the further application of these methods ill result in the analysis of many others.

It was concluded that the use of colorimetric methods for the assay of sugar phosphates in mixtures is of limited value in such work. An exception was the orcinol assay method for pentose. The phosphorus compounds were not separated sufficiently on Dowex 1chloride columns to analyze them quantitatively from TP assay, UV absorbancy measurements, and colorimetric sugar analyses on the



49






50


eluted fractions. The eluates from Dovex 1-chloride columns could be rechromatographed on finer mesh Dowex 1-formate columns, however, resulting in further separation of phosphorus compounds. The compounds thus separated appeared to be in almost pure form, except for the hexose monophosphates, which were not separated from each other under the test conditions. This method of rechromatography of chloridecontaining eluates was unfortunately limited in its usefulness to eluates 0.02 N or less in chloride ion, because of the swamping of the phosphorus compounds at high chloride concentrations. Slower elution of the Dovex 1-chloride column with smaller amounts of chloride eluent may overcome this difficulty.

The use of Dowex 1-formate columns to separate directly the

tissue extracts, without prior separation on Dovex 1-chloride offers promise. The ammonium and formate ions, unlike the chloride ion, can be essentially removed from the eluates. Dovex 1-formate rechromatography of selected portions of these eluates, using various amonium formate buffers, would be expected to provide further separation.

The enzymatic analyses proved to be useful when dealing with

eluates of varying degree of purity. It may prove practical, as well as expedient, to perform such analyses on the five relatively mixed eluates from Dowex 1-chloride columns. The firefly lantern extract (luciferase) method was found to be an extremely sensitive method for measuring ATP in plant tissue.

The concentrations of the phosphorus compounds identified in acid extracts of corn roots should be confirmed by additional tests.


--7- It 111---W


















SU1iA.RY


Methods were described for the separation and analysis of

glycolytic intermediates in etiolated corn seedling roots, with the use of ion exchange column chromatography. The following compounds were identified: inorganic phosphate, glucose-l-phosphate, glucose6-)hosphate, fructose-6-phosphate, monophosphoglyceric acid, adenosine diphosphate, adenosine triphosphate, and uridine diphosphate glucose.


51





- r- *:.f~' ~' ~


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A. -. -








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55


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.,~w- -


iI


3IOGBAPHICAL SKMTCH


Byron H. Wise as born 12 February 1925 in Gainesville, Floride. He graduated from P. K. Yonge Iaboratory School in 1942.

He attended the University of Florida from 1942 to 1944. After service in the Arr, he re-entered the University in 1946, and received the Degree of Bachelor of Science, with Honors, in 1949. His major subjects were Biology, Chemistry, and Psychology.

He received the Degree of Master of Science in Agriculture in 1953 from the University of Florida. His major subject was Botany, specializing in TaxononW.

He took two years of graduate vork at Washington University, St. Louis, Missouri. For four years he was an industrial research chemist.

In 1959 he started a graduate program in the Department of Botany, University of Florida, majoring in Plant Physiology. He is a candidate for the Degree of Doctor of Philosophy, to be awarded in December, 1962.

He is a member of the following honorary societies: Phi Eta Sigpa, Gas=n Siena Epsilon, Phi Sigma, and Phi Kappa Phi.

He is married to the former Winnie Sue Moss.


58















This dissertation was prepared under the direction of the chairman of the candidate's supervisory committee and has been approved by all members of that committee. It was submitted to the Dean of the College of Agriculture and to the Graduate Council, and was approved as partial fulfillment of the requirements for the degree of Doctor of Philosophy. December, 1962




Dean, College of Agricultii'





Dean, Graduate School Supervisory Committee: Chairman







62I- e e




Full Text

PAGE 1

ACID-SOLUBLE PHOSPHATE COMPOUNDS OF CORN ROOTS By BYRON HOOPER WISE A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA December, 1962

PAGE 2

The writer expresses his appreciation to Dr. T. E. Hxunphreys for his thou^tful guidance during the coiurse of this research; to Dr. G. R. Hoggle for encouragement and financial support throu^ the Depcurtment of Botany during the entire doctoral program; to Dr. A. T. Wallace for encouraging the writer to undertake the program; to Drs. R. D. Powell and T. W. Steams for serving on the comalttee. Special credit is due my wife, Winnie Sue Wise, ^Aio made this woz^ possible. 11

PAGE 3

aiossAM Pt pbosphorus R5P: riljose5 -phosphate PPt pyrophosphate OIP I glucose -l-phosphate IP: inorganic phosphorus G6P: glucose -6-i^osphate TP: total phosphorus PIP J fructose-l-phosphate 03P: glyceraldehyde-3-idiosphate P6P: fructose -6-phosphate MPO: monophosphoglycerate FDP: fructose -l,6-dipho8phate SPQk: 2 -phosphogly cerate HKP: hexose monophosphate yKAt 3-phosphoglycerate HDP: hexose diphosphate SPG: 2,3-dlphosphoglycerate IPS or DFBH: oxidized or reduced dlphosphopyrldlne nucleotide TFN or TFHH: oxidised or reduced trlphosphopyrldlne nucleotide AMP, ADP, ATP: adenosine mono-, dl-, triphosphates CMP, CDP, OTP: cytldlne mono-, dl-> triphosphates GBff, CH3P, (TOP: guanoslne mono-, dl-, triphosphates UMP, UIP, inPi uridine mono-, dl-, trlphosj^tes IBP, rrP: InoBlne dl-, triphosphates UPPO: uridine diphosphate glucose 111

PAGE 4

TABLE OF COHTEHTS Page ACKNOWIZDGEMEKT H GLOSSARY iij LIST OF TABLES vl LIST OF FIGURES vii STATEMENT OF PROBLEM 1 REVIEW OF LITERATURE 2 MATERIALS AND METHODS l5 Plant Materials Preparation of Extracts Preparation of Ion Tg-gnhftngo Colxams Colvmn Chronatography Analytical Methods Phosphorus (TP, IP, T-ndn P) ass^ Assays for sugar phosphates Assay for gly cerates Enzymatic assays for phosphorus costpounds G6P and F6P Uridine dijiiosphate glucose (UDPG) Microdetermiaation of ATP with firefly lantern extract Analysis from UV absorbancy measxireiaBnts

PAGE 5

Pa«e RESOIIPS 26 Sovex 1-Chlorlde Chrooatografby of Kncnm Ccsqpouads Sowez l-Fornote Chrcnatograpfay of KhovQ Conipoijnds Dovex l-Chlorlde Chronatography of Corn Root Tip Extracts Dovex l-Foroate Chromatography of Eluates from Dovex I — Chloride Separatlcms of Com Boot Tip Extracts Direct Dovex 1-Ponnate Chromatography of Com Root Tip Extracts AZP Assay of Com Root Tip Extracts Phosphosnos Compounds Identified in the Root of the Etiolated Com Seedling D33CUSSIQH ^9 SUMftRY 51 HTHTiTOgRAPffif 52 BIOGRAPHICAL SKETCH 58 T

PAGE 6

LIST OF TABLES Table Page 1. PHOSPHORUS COHTENT OF A TRICHLOROACETIC ACID EXTRACT OF 63-2 GRAMS OF CORN ROOT TIPS, BEFORE AHD AFTER TREATMEHT WITH DOWEX 50 29 2 . PHOSPHORUS CCMjTENT OF SEVERAL TRICHLOROACETIC ACID EXTRACTS OF CORN HOOT TIPS, AFTER TREATMOn' WITH DOWEX 50 30 3. TP RECOVERY IN THE DOWEX 1-PORMATE CHROMATOGRAPHY OF THE 0.01 N IKl SECTICMI FROM DOWEX 1 -CHLORITE CHROMATOGRAPHY OF FOUR CORN ROOT TIP EXTRACTS ... 34 k. ABSORBANCY RATIOS AT 25O, 260 , 280 MJ kO 5 . raOSPHATE COMPOIHDS OF CORN ROOT TIPS 48 vi

PAGE 7

LIST OF FIGUBES Figure . Page 1-h DOWEX 1-CHLORIDE CHROMATOGRAPHY OF TRICHLOROACETIC ACID EXTRACTS OF CORN ROOT TIPS 3I 5 DOWEX 1 -FORMATE CHROMATOGRAPHY OF THE WEAK ACID PHOSPHATE COMPOUNDS FROM A TRICHLOROACETIC ACID EXTRACT OF I8.6 GRA^B OF CORK HOOT TIPS 32 6 DOWEX 1 -FORMATE CHROMATOGRAPHY OF THE SUGAR MONOPHOSPHATES (PEAK 1) FROM AH EXTRACT OF 38.3 GRAMS OF CORN HOOT TIPS 36 7 DOWEX 1-PORMATE CHRCmTOGRAPHY OF THE 0.02 K lEl SECTION OF A TRICHLOROACETIC ACID EXTRACT OF 57.1 GRAMS OF CORN ROOT TIPS 38 8 DIRECT DOWEX 1-FORMATE CHROMATOGRAPHY OF A TRICHLOROACETIC ACID EXTRACT OF 51 .8 GRAMS OF CORN ROOT TIPS 1^1 9 DIRECT DOWEX 1-FORMATE CHROMATOGRAPHY OF A TRICHLOROACETIC ACID EXTRACT OF 51 .8 GRAMS OF CORN SOOT TIPS 1^2 10 DIRECT DOWEX 1-FORMATE CHROMATOGRAPHY OF A TRICHLOROACETIC ACID EXTRACT OF 71-9 GRAhB OF CORN ROOT TIPS 43 11 ULTRAVIOIBT SFKTTRA OF THE POOLED TP PEAKS FROM DIRECT DOWEX 1-PORMATE CHROMATOGRAPHY OF TRICHLOROACETIC ACID EXTRACTS OF CORN ROOTS .... h6 vii

PAGE 8

aEATEMEHT OF THE PROBLEM The glycolytic sequence represents a crucial segment in the intermediary metaholism of the plant and animal cell. The operation of the glycolytic system can he expected to he influenced hy the levels of each substrate and hy the activity of each enzyme, in the series . The presence in hi^er plants of most of the glycolytic intermediates is documented. Hovever, very little is knovn about either the concentrations of these compounds, and their aissociated coenzymes, in normal plants, or their changes in relation to physiological changes. The reasons for this i)aucity of information can be traced to the severe technical difficulties encountered by various workers in applying quantitative analytical methods to plant materials, particxilarly the higher plants. The object of this investigation is to develop methods to determine the levels of various glycolytic intenaediates in plant tissue (etiolated com seedling roots) using ion exchange column chromatography as the basic means of separation. 1

PAGE 9

BEVIStf OF IiHEBATUBE The methods einployed In this investigation are based on the separation of acid-soluble, phosphorus -containing compounds of plant tissue prior to their quantitative determination by various methods. The study involves a broad general siirvey of methods, including phosphorus assay, assay of sugar phosphates, assay of associated coenzymes, extraction methods, various types of ion exchange chromatography, and, finally, a review of the methods which have been used to stiody phosphorus con^raunds in plants. The following review is presented in the above sequence. It will be noted that most of the work In the field has been done with animal tissues. It will also be noted that there is some overlapping in presenting the pertinent articles. This occurs because it was thou^t that a review stressing methods would be more iiseful if the recent work involving icm. exchange chromatography be presented with a modicum of detail. Many methods have been described to assay jiiosphorus in biological material. Most procedures depend on the intense blue color \jhich is produced ^iien the phosphomolybdate complex is reduced by various agents. The color intensity is measured in a photoelectric colorimeter against a standard series of known phosphate concentrations. One of the earliest methods (15) utilized hydroquinone for reduction; however, the color iiias evanescent. Fiske and SubbaRow 2

PAGE 10

3 (30) discussed several factors affecting accurate phosphorus deterndnatiooB. Variations of their method, involving reduction by aminonaphtholsulfonic acid in the presence of E^Ok, are still popular (13). The oethod is reportedly insensitive to a 30^ variation in acid, BolyMate, or reducii^ agent ccmcentration . Berenblum and Chain (17) described a oethod in vhlch the idiosphcaoolybdic acid is reduced to the blue cotqplex by shaking with a mixture of butyl alcohol and stannous chloride and separating the blue alcoholic layer. The nethod is said to be insensitive to tenfold increases of the reagents. The Gomori (31) aK)dification uses the photographic developer Slon (nethyl-p-amlnophenol sulfate) for reduction. In general^ is detemined by neasurement of color production after 10 to I5 min of developnent; TP is similarly determined after vet-ashing the sample in HgSOl^. nie estimatlc^ of IP in the presence of ATP with a mjnlmiun of iK>Iybdate-cat£dyzed hydroI;7Sis of ATP is described by Marsh (5^)« The excess molybdate is reuDved as a citrate complex after the extraction of phosphomolybdate by butanol. Alternately^ the IP can be precipitated by CaCl2-Ca(GH)2 to remove various labile ^osphate esters, such as creatine phosphate and ATP. Several colorlmetric methods \ised. for estimation of siogars and sugar phosphates are described by Ashvell (10). The enthrone, cysteine -carbazole, and orcinol procedures (lO) are colorimetric assays for hexose, fructose, pentose, and their phosphate derivatives. Two coBQXJunds as similar as ribose-3-phosphate and R5P may be differentiated under carefully controlled heating ccajditions ( 7 ) . In * See glossary.

PAGE 11

k practice, the results have been equivocal (lO) except \^re considerable purification vas attained prior to coloriuKtry. Hovever, Baxtlett (13) has reported the determination of ketohexose phosphate with cysteine -carbazole, and pentose jAiosidiate with orcinol, in flections containing a mixture of these esters. Further, Bartlett has claimed quantitative assay of g6P by svibtracting the cysteine -carbazole result from the anthrone result obtained on assaying a mixture of g6p and F6P. Hoirever, Helbert and Browx (35) found that color production in the anthrone assay varied from hexose to bexose . GIP has been estimated in mixtures of bexose phosphates "by metisuring the increase in IP SLtter 7 lain at 100 C in 1 H HCl (U?). Eiiaymatic methods are available for the estimtion of sugar phosphates. FDP can be assayed by the method of Slater (70), using the rabbit muscle fraction A of Backer (63), \*iich contains aldolase, triosephosphate isomerase and d -glycerol phosphate dehydrogenase. The change in optical density at 3^*0 mu due to oxidation of added DPHH is used to calculate FDP. The method measures the sum of MP, 63P, and mP. F6P can also be estimated by SUter's procedure (70). When Backer's (63) rabbit muscle flection B (containing phospii
PAGE 12

5 according to the procedure of SlAter (TO), by laeasuring the change of fluorescence of TPMH, and, alternatively, measured ATP l>y adding glucose and hexokinase to the reaction mixture used for the estiiation of g6P. Komherg (1*0) deterained ATP hy this method (spectrophotometrically) . Ochoa et al. (6o) assayed pyruvate spectrophotometrically in the presence of lactic dehydrogenase and EPHH. Seraydarian et al . (69) adapted fluorometric methods to the detensLnation in naiscle extracts of millimicromolar quantities of (a) FDP plus triose phosphates and (b) hezose monophosphates. The two groups were not separated; the authors felt that the two groups were ia^rtant in representing glycolytic intermediates of similar phosphate coaipoBition. The levels of ad enine nucleotides, especially ATP, are pewticularly itiqwrtant in biochemistry of animl and plant tissues. ATP has been estimated by the method of Sljrter ( 70) and the micro nethod of Seraydarian (69). McELroy's finding (1*7) that added ATP caused li^t production in the darkened extracts of firefly lanterns opened the possibility of using the phenomenoa to assay ATP. Further experiments by the McELroy group (kS) showed that the production of li^t in firefly extracts (FFE) depends on a heat-labile enzyme (luciferase), a heat-stable, yellow-green luminescent c(M5>ound (luciferin), an inorganic ion (Mg^^^ Co"^, or Ifa"^), oxygen, and ATP. A partial purification of the FFE (49) resulted in the removal of uQrokinase and adenosinetriphosphatase , and revealed the specificity of the luciferin-luciferase reaction to ATP. Strehler and Totter (71) described the appUcaticn of the system to the estiimtion of mixtures of ADP and ATP.

PAGE 13

6 The FFE assay for ATP has been used in Investigations on an1m1 tissues, especially the nervous system. Single frog sciatic nerves (ca. 25 mg) were heated In boiling 'Trls* buffer for 15 sec, chilled In ice, homogenized in the cold, shaken vlth CC1|^, and centrifuged. ATP vas estimated on the clear supernatant, using controls also containing CClj^ (33 )• Veiner (73) en^iloyed special procediires to minimize the rapid breakdown of labile ];^osphate cos^unds vhlch occurs in the central nervous system of rats killed by decapitation. The animals vere frozen rapidly by immersion in liquid oxygen, the baraln was chipped out and extracted in cold perchloric acid, and the acid ms precipitated vlth KOH. The change in optical density at 251 and 265 mu upon additicxi of adenylic acid deaminase uas assumed to be equivalent to AMP in the Bajsple. H/oklnase vas then added, and the change in OJ)^^ ws proportional to one half the amoimt of ADP in the saarple . Blanks and knovn mixtures of nucleotides vere run vlth each series, and correcticMis were made for sll^t non-specific change in ODg^jL, due to addition of enzymes. Chens (22), working vlth trlchloroewetic acid extracts and perchloric acid extracts ( 1 ) of nerve trunks, coupled the following enzyme systems, each folloved by a heat inactlvatloo step: (a) Pyrophosphorolysis of ATP in presence of sulfurylase (6U) and pyrophosphatase (kl) (b) Phosphorylation of AEP to ATP vith creatine phosphoklnase ik2) (c) The final assay of AIP (phosphorylated Alff) with luclf erase (71). Mlnard and Davis (58) used even more exacting msthods of extraction of rat brains than did Weiner ( 73) for the separation of nucleotides, especially ATP. They immersed the rat suddenly in

PAGE 14

7 liquid nitrogen for several minutes. The brain vas chipped out, collected in a mixture of Dry-Ice and acetone, q\iickly pvilverized, and extracted in trichloroacetic acid at 0 C. Chroxnatographic separation of nucleotides was hased on the method of Hurlbert et al. (38). Phosphorus measurements and 0^260 ^^""'"SS were made on each fraction to calculate the total nucleotide concentration. There vas no correction for fonnate, vhich absorbs strongly in the ultraviolet region. The positions of adenine, guanine, and uridine nucleotides were determined by conqparison with chromatograms of known nucleotides. The peak fractions were pooled, subjected to hydrolysis in 1 H HCl, and the individual bases were identified by paper chrcanatography . Portions of the eluate fractions containing Alff,. AII»; and ATP were passed throu^^i columns of Dowex 50 resin (hydrogen form) to remove ammonium ion, evaporated to dryness in vacuo to remove formic acid, and hydrolyzed. Con^Kments of the hydrolyzates were separated with a Dowex 1-chloride column developed with 2 -ami no-2 -methyl -1 , 3 -propanediol and ICl. This system can separate adenine, guanine, Clff, UMP, and AMP. Bishop et al. ( 19) separated the nucleotides in trichloroacetic acid extracts of whole blood, according to Hurlbert et al. (38); but non-gradient elution was used. The material in each peak was evaporated to dryness on a steam bath, and the free purines and pyrimidines liberated by hydrolysis for 1 hour at 72 C in 72^ perchloric acid. Neutralized solutions were chromatographed in the isopropanol-HCl system of Wyatt (Tk) followed by the butanol-KHj^^OH system of Markham and Smith (51)The bases were eluted with dilute BCl, and their spectra determined . Cohn (23) introduced ion exchange column chromatography into the

PAGE 15

8 nucleotide fieldAdenylic, uridylic, guanylic and cytidyllc acids were eluted from Doxrex 1-chloride polystyrene anion exchange resin columns by dilute HCl-HaCl solutions in a sequence rou^ily predictable from pK values. Cbromatograms were prepared by measuring OD^^q of each fraction collected and plotting these values against the volume eluted. Cohn and Carter (24) similarly eluted AMP, ADP, and ATP. Paper chromatograms were developed on material from peak tubes, using an isoanyl alcohol-disodlum phosphate mixture and the nucleotides detected by ultraviolet fluorescence . Horecker and Sinymiotis (36) reported a partial separation of R5P and a similar substance later found to be ribulose-5-phosphate on a Dowex 1-f ornate column by elution with 0.1 M formate bxiffer. This was a pioneer experiment in the elucidation of the pentose i>hosphate shunt. The pentose phosphates were produced vhen a pvirified yeast enzyme was mixed with 6-phosphogluconate . Benson et e^. ( 16) separated P6P from FDP on a Dowex 1-chloride column eluted with 0 .1 M HaCl . Khym and Cohn ( 39) reported the separation of synthetic mixtures of sugar monophosphates (GIP, G6P, F6iP,R5P) by forming borate complexes simultaneously with elution from Dowex 1-chloride column with four chloride solutions. Hexose phosphates were estimated by the enthrone method (59), B5P by the orcinol method (2l), and phosjiiorus was assayed according to Fiske and SubbaEow (30). Goodman, Benson and Calvin (32) used the above techniques of Khym and Cohn (39); Benson et al. (16), and Horecker and Smymiotis (36) to study photosynthetic products in ethanol extracts of the al^, Scenedesimis A synthetic test mixture of 76P, TDP, and 3PGA, aad a

PAGE 16

9 mixture of 3PGA, FDP, and Ribulose-1, 5-dlphosphate was separated on Dowex 1-chloride "by elution with a dilute HaCl-HCl solution. They also separated FIP, YSP , and G6P hy elution with 0.1 M HaQBl^O^ from a column converted to the borate form by eluting a Dowex 1-chloride column with Ha^i^Orj' Finally, they chromatographed a simple synthetic mixture of the above sugar phosjiiates mixed with an extract from algae grown in the presence of P-' . Radioactivity was found in peaks corresponding to various esters, indicating the natural incorporation of phosphorus in those compounds by the algae. Aisenberg ( 2 ) used Dowex 1-chloride columns mainly to remove free sugars from the glycolytic intermediates in the acid-soluble fraction of high-speed supernatant of brain extract. The free sugars were washed thixiu^ the column with water, the esters were eluted batchwise with 0.1 N HCl, the eliiate was concentrated by lyophilization, and g6P, F6P, G3P and hexose diphosphate were enzymatically assayed ( 25) • Ber^vist and Deutsch (18) separated synthetic mixtures of mono-, diand triphosphates of adenine, guanine, and uridine by applying seven successively increasing concentrations of formate buffer. OD250 °^ each fraction was plotted against volume through the column and identification of each peak was made by comparing its UV spectrum with spectra of known nucleotides. Diedrich and Anderson (28) used the method of Goodman et al. (32) to separate galactose-l-phosphate from other common hexose monophosphates in a synthetic mixture. They described two raethods to remove the troublesome borate ion from column eluates. They found, as had Helbert and Brown (35), that the color in the anthrone assay (65)

PAGE 17

10 varied from hexose to hexose, they coii5)uted valiies from each parent sugar. The method they generally used to monitor fractions was based on Dische et al. (29). This modification enabled them to distinguish i)entose from hexose in mixtures. Diedrich and Anderson (27) used similar methods in studying the appearance of galactose. Trichloroacetic acid extracts were resolved into several anthrone -positive peaks, each of vAiich was revealed by paper chromatograpiiy to contain several unidentified conqpounds. The fractions of the first two peaks were pooled, and tested enzymatically for galactose-l-phosphate . Tiselius et ( 9 ) discussed some limitations of stepwise elution and introduced a new general procedure for 'gradient elution " of mixtures of carbohydrates. Gradient elution has since been successfully applied to mixtures of organic acids, amino acids, peptides, proteins, and nucleotides. I^shmanan and Lieberman (43) stated the sidvantages of using concave gradient elution, in lAiich the rate of change of eluent concentration increases with the concentration. The more easily eluted substances are spread apart in the chromatogram, and, since the concentration toward the ejad of the chromatogram increases rapidly, the more tightly bound substances are eluted without undue delay. This system also produces narrow peaks with a minimum of tailing. Pontis and Blumson (62) used concave gradient elution to separate a synthetic mixture of nucleotides on an ion exchange resin. Bock and Han-Sing Ling (20) described systems generating several types of elution gradients. Hurlbert et aL (38 ) described a system of "extended gradient elution." The reservoir, containing the concentrated eluent, and

PAGE 18

the olxiag vessel, initially containing water, were designed so that tbe contents of the reservoir could he changed at intervals. Several aoBaQai\im foxnate huffers nere used to separate the nucleotides in synthetic Bixttires. The nucleotides in a perchloric acid extract of rat liver were similarly sepeurated. Martonosi (55) used extended gradient elutica (38), a Dovex 1-colum (hicarhonate form), and KBCO^ eluflixt sol\xtiGiis to separate a synthetic nlxture containing Alff, AIS*, ATP, nra, IP, THI, EP, HP, and HP. Bicarbonate was removed from eluate fiactions hy neutrallxing with perchloric acid, followed by eaitrifugatioai of the potassium perchlorate precipitate. A d enine and «i^«»n^nA nucleotides were assayed from ODg^Q values; DPH, IP, TFH, PP, US', and ISP were assayed by IP and TP determination. ' Hills (^7) used extexkded gradient formate chromatography to separate several nuclaotides in red blood cells. Monitoring vas done liy measuring ODg^ of each fraction. Humerous chromatograms of known niicleotides were run for coapairison . Several nxicleotides were quantitatively esttmsted by using the appropriate extinction coefficients after they were first identified by coB?)arljig their absorbancy ratios in the region from 2^5 to 29O mu with the ratios of know nucleotides and derivatives. (61) Wade ( 7^ devised a complicated system, «BQ>loying p\m^ which operated autoititically in response to a pB-sensing device, to separate various synthetic mixt\ure8 of phosidiate esters. The system was deaigped to cban^e the pfi over a much wider range than could be obtained by changing buffier concentration. Bartlett (12) xeported a very extensive investigation of glycolytic intexnediates and co-enzymes in red blood cells. This work

PAGE 19

12 was based cm anion exchange chromatography, but many related techniques were employed. Studies on phosjAiorylated coapouads In plants to the year 1952 are reviewed by Albaum (k). In the earliest studies, the acidsoluble fraction was assayed for inorganic jdiosphoruB and organic phosphorus. ATP was measured by determining the increase in IP after hyirolyzing the acid extracts for 7 minutes at 100 C in 1 N flCl; it was assumed that all phosphorus labile under these conditions is AZP phosi*iorus . In 191^3 Lepage and Umbreit ( presented a system to determine specific P-coii5>oundB in synthetic mixtures of pure con^xjunds. This method was based on the finding that DP, ASP, ADP, PGA, and FDP form soluble barium salts under the same conditions. They vere able to apply this method to trichloroacetic acid extracts of TfalobacilluB thioxidans by carefully controlling the quantity of tissue and reagents. IP ws determined directly. PGA vas estimted by a colorimetric reaction then thoui^it to be specific . PBBP and P6P trere estimated by a colorinetric method. GIP i&s estlaated as "seven minute phospiiorus . " EPH was estimated from the concentration of nicotinamldfi . AMP -vbls estimated from nitrogen and rlbose deterndnations, after correcting for BEK. ATP and AIX> were measured by determining total nitrogen as an index of purine present, pentose assay, and ratio of labile phosphorus to total orffmlc phosiijorus, after deducting the phosphorus resulting trovi other coB5»unds present. Correction had to be made for phosphorus hydrolyxed from FEP (about 27^). The remaining phosphorus was assuned to be g6P. Despite the large number of assuaq?tions and corrections oade by LePtege and

PAGE 20

13 Umbreit, they reportedly accounted for over 90^ of the acid-soluble jdioephomifl . Since this general method worked in bacteria (and »^^^n^^^^ tissues), Albaum and Unibreit ( 8 ) attenqpted to apply it to higher plants (oat seedlings). Large amounts of non-specific absorption resulting from the presence of various polysaccharides, frustrated theri efforts to apply colorimetric sugiar assays. Also, after applying all the corrections, much jdiosphorus still could not be accounted for. It was known that cereals ccaitfiin quantities of phytin but correction for phytin still left some jiios^oiMS unaccounted for. Albaum and Ogur ( 5 ) tried to isolate ATP from oats by traditional methods of fractionation with bariim and mercviry. Although polysaccharide could be removed from animal tissues by precipitation with ethanol, this was not true of the oat extracts; polysaccharides were carried along in each precipitate and each supernatant. Also, barium phytate was precipitated under almost the same conditions as ATP. Finally, there was much unknown material absorbing nonspecifically in those parts of the ultraviolet spectrum where the absorption of nucleotides was meixiraum. Albaum et al. solved the problem neatly by turning to a non-cereal source of plant material, the mung bean. This material yielded ATP of about 70^ purity. The Albaum group ( 6 ) also tried to apply the fractionation to Euglena . The presence of inorganic jdiosphate, pyrophosphate, metaphosphate , AMP, ADP, ATP, EEN, CIP, F6P, PGA, and riboflavin phosphate was reported. Ifarre et al.(53) studied the effects of auxin treatment and pollination on levels of ATP and some glycolytic intermediates in tomato ovaries, by the methods of LePage and Umbreit (^+6).

PAGE 21

Ik Most of the early fractionation procedures required large amounts of plant material. In the isolation of ATP from mung beans 5 to ID pounds were used. Later colunm chromatography and paper chromatogi-aphy were used with smaller samples of material. Albavuii(U), working with mung bean seedlings, prepared trichloroacetic acid extracts from 3 g of tissue, precipitated the P compounds ^^ith ethanol and barium, and precipitated barium with H2SO4. The solution was poured through a small column of Dowex 1-chloride, and eluted stepwise with five increasingly concentrated chloride solutions, displacing adenosine, adenine, AMP, AIP, and ATP in succession. laughman and Martin (kk) separated acid-soluble organic phosphorus compounds from roots after short periods of absorption of by young barley plants. Ejrtraction by 0.2 K HCl, O.5 H trichloroacetic acid, or 80^ ethanol gave similar results. Separation was perfomEd by paper chromatography by the method of Hanes and Isherwood (34). Five ra^oactive spots \rcre resolved by a butanol-water-picric acid system. Each had the appearance of a single compound, but each was composed of two or more substances, which could be separated by the use of other solvent systems. Unkno\m radioactive compounds were mixed with the pure unlabelled forms of suspected compounds. Chromatograms were then run in solvent systems separating the known substance from others. Active areas were compared with those developed by a molybdate spray, reveaaed under UV light. Several investigations have dealt vrLth the assay of phosphate esters in the fruit and seedling of pea. Pi sum sativum L. Rowan et al. (68) reported changes in levels of ADP, ATP, HMP, and HEP in ^t^rotloric acid extracts of pea fruits at various stages of maturation, by

PAGE 22

15 Slater's procedure (70). There \ra.s a possibility of interference in the determination of AXP by OTP and GffP (67), but, since paper chromatograiQS of the nucleotides shoved the latter materials to be present in small amounts, no correction vas made. Rovan (66) extended the investigation to the quantitative determination of uridine mono-, di-, and triphosphates, as well as AIF and ^QT. In this work a large aioount of material was used (2^0 g to 2 kg) . Marre and Forti (52) extracted stem sections of seven-day-old pea seedlings in perchloric acid. The extract vas treated according to the method of Crane and Lipoenn (26) vlth "Borlte A" charcoal, \iblch. absorbs nucleotides but not sugar phosphates. The nucleotide-cc»italnlng Borlte A vas filtered, heated in a boiling vater bath for 10 minutes in 1 N BCl, and IP vas assayed. Crane and LLpmann (26) extended this procedure to measure AIX> and AZP labile phosphorus, but other nucleotides mi^t be present in such an extract, ^diich vould result in hl^Ji readings.

PAGE 23

MATERIALS MD MBfHDDS Plant Materials Dried kernels of hybrid seed com ( Zea mays L., var. Fjnk's and 6-7'*^0) were rinsed thoronf^ly, and soaked vith aeration In tap VBter for 2k hr. The kernels yere rinsed again and then placed individually on trays lined with thoroughly wetted filter paper. Each tray was covered and the kernels vere allowed to germinate in the dark at 25 C for T2 hr. The distal 2 cm was cut from the primary root of each seedling and collected in ice-cold distilled water. The root tips were filtered in a Buchner funnel, washed once with ice-cold water, gently spi^ad on filter paper to remove adhering water, and weighed to the nearest 0.1 g. A typical harvest of 65O to 700 root tips yielded ahout 20 g of tissue. Preparaticai of Extracts The wei£^d root tips were thorou^Jily groimd for about 3 min in 10^ trichloroacetic acid (l ml/g of tissue) in an Ice-cold mortar. The resulting slurry was centrifuged, and the supernatant liquid was decanted. The residue was suspended in 5?6 trichloroacetic acid (1 ml/g) and centrifuged. The combined supernatant fractions were retained; the residue was discarded. All manipulations to this point were performed at O-5 C. The trichloroacetic acid was reiaoved "by four extractions vith two volumes of cold ether; the pH of the extract was then about 3.5. A thin stream of nitrogen was bubbled through the 16

PAGE 24

17 extract until tbe ethereal odor vas not discernible, and ^ H HHi^OH ymB added to pH 6.8-7.0 This extract vas passed throu|^ a Dovex 30 colian, and followed by ^0 ml of water; tbe coabined filtrate vas neutralized to pH 6.8-7.0. One to four extracts were used in chronatography. The extracts could be stored several weeks at -20 C. Prepeuratlon of Ion Exchanae Coluims TrinethylaiarBonium polystyrene ( "Dowex" ) resins (Dov Chenical Co . ) were obtained in a purified form (Bio-Rad laboratories, Richnond, Cal l f . ) . Tvo types of anion exchange resins and one type of cation exchange resin were used. One anicm exchange resin, AG I-YB, ^0-100 nesh, was suspended in water and the extremely fine particles were decanted. Fourteen-cm resin beds were formed in 1 X 30-cm glass chromatographic tubes with sintered glass retainer's (Erail Greiner Co.)« Uniformity of the many colvuans prepared during these investigations was approached by stirring thorou^Jily the thin resin slurry Imaediately before pouring the columa, and repeatedly inverting the colunm while adjusting the resin bed to the desired l^igth. The resin columns were each capped by a small plug of washed glass wool, and eluted suxicessively with two bed volumes of 88^ formic acid (38), three volumes of water, three volumes of 1 H HHi^Cl, and, finally, with water until the filtrate had the same pH (5.5-6.0) as the distilled water used, and was also chloride -free . Chrcnatographic coluons prepared in this manner are subsequently referred to as "Dowex 1-chloride" columns. The other anion exchange columis, containing AG 1-X8, 200-400 Bsh, were prepared as follows. The extreme fines were decanted and the amorphous particles were removed with a medicine dropper. One X

PAGE 25

18 l4-cm resin Ijeds were povired as descriTaed above aad eluted with three "bed volumes of OSfft fomic acid, four volumes of 5 H anmc»ium formate (until the filtrate was chloride -free), and with an excess of water. Such resin colunms are referred to herein as "Dowex 1-formate" coluims. The cation exchange columns were prepared from AG 50tf-X8, 100-200 mesh. Three-cm beds were poured in 1 X 30-cm tubes. The resin was eluted with 30 ml of 2 H HCl, with water until the filtrate was chloride -free and with an excess of water. ColuHP Chromatography Solutions containing anions to be separated by ion exchange colum chromatography \rere passed throu^ Dowex 1-formate coliams at flow rates of about 3.0 ml/min. Solvent flow was mintalned by two to four potaads of air pressure from a low pressure regulator (Matheson Co.) and adjusted by a teflon-glass needle valve (Emil Greiner Co.). Ccnnecticais were made with silicone rubber tubing Type HPR (Ronsil Co., Little Palls, N.J.) after it was found that "tygcMi" tubing introduced significant contamination in colorlmetric analyses of the eluate fractions. The fractions were collected by an automatic fraction collector (Research Specialties Co.), with a volumetric siphon; graduated test tubes were used until the reliability of a siphon was established. Twenty-ml fractions were collected from Dowex 1-chloride colxomns and 10-ml fractions were collected frcaa Dowex 1-formate colujnas. Each fraction was assayed for TP, and chronBtograms were always prepared to reveal the eluticm positions of P-containing cosipounds . The anions that were adsorbed on Dowex l-chloride columns were eluted at about 2.8 ml/min by 200-400 ml of each of the following

PAGE 26

19 solutions (13): 0.01 N HCl, 0.02 N HCl; 0.1 K KHj^Clj 0.2 K NHj^Cl, 0.5 K IIHi^Cl. In prelimineiry experiments, the fractions ohtained with each of the five chloride eluent solutions from a Dowex 1-chloride column, containing compounds of similar resin-binding capacity, were pooled, passed throu^ a Dciwex 1-formate column, and the anions were eluted by formic acid or anmonium formate buffers. In later experiments the Dowex 1-chloride column eluate was collected in five batches corresponding to the five eluent solutions. Each batch was neutralized with KHi^OH to pH 6.8-7.0, and was ftored at -20 C until chromatographed with a Dowex 1-formate column . Dowex 1-formate columns were also used to chromatograph directly whole trichloroacetic acid root tip extracts without prior sepajration on Djwbx 1-chloride columns . The fractions in a TP peak were often pooled, i)as6ed through Dowex 50 (hydrogen form) to remove ammonium ion, extracted several times with ether to remove formic acid, neutralized, and retained for analysis. Dowex 1-forraate columns were eluted at about 1.0 ml/mln with linear or concave gradients of formic acid or aunmonium formate buffer. Concentration gradients were obtained with systems diagrammed by Bock and Ling (20). A linear gradient was obtained when the crosssectional areas of the two eluent-containing vessels were the same. Concave gradients were obtained when the area (A^) of the mixing vessel was greater than that of the reservoir (A2)The ratio, ^ = 0.6, was selected for use in these experiments (62). However, Al linear gradients were used more often than concave gradients after preliminary experiments with the latter.

PAGE 27

20 Various synthetic test mixtures of known phosphate coiirpounds were also chromatographed by the same procedure with Dowex 1-chloride or Dowex 1-fonaate systems for comparison with chromatograms of plant extracts . / Analytical Methods Fhosphonis (TP, IP, T-mln P) assay . Phosphorus was aesayed colorimetrically by taeasuring the blue phosphomolybdate coniplex. The following modification of the Gomori (31) method permitted reliable and relatively convenient assay of large nuniberB of sanrples. For TP aissay, the sample was wet -ashed by heating for 3 hr in an oven at 170-175 C in a 10 X l80-am test tube calibrated at 10 ml and containing 1.0 ml of 5 H H2S0i^ and a glass bead. Two drops of 305^ hydrogen peroxide were sidded, and heating was continued for an additional 2 hr. Three drops of 5^ urea were then added and heating continued at 100 C for 3 br. One ml of water was added and heating continued at I2O-I3O C for 1 hr. After the tube had cooled, two ml of 6.2% Na2Mo04*2H20 in 5 N H2S0i; were added, followed by 1.0 ml of 1^ Elon (31) (Eastman Co.) in 3^3 HaHSO^. The color formed was read, after 10 to 60 min development, in a photoelectric colorimeter (Klett Co.) using filter #64. Calibration curves were prepared using an inorganic phosphate standard solution. IP \ra,s assayed si milar ly, but without heating. The temperature was kept belov 22 C, and the sample was diluted before axidition pf acid, to minimize hydrolysis of labile phosphorus compounds (27). Acid-labile, or "7-min" phosphorus was estimated by the increase in IP after 7 min at 100 C in 1 H IKl . Assays for su^^rar nhosiAates . Hexose was assayed by a modification

PAGE 28

21 of the ajithrone method (10). Two-ml samples were layered over k.O ml of 0.2^0 anthrone in 95^^ H^SOj^ while chilled in an ice bath. The mixture was shaken vigorously, heated for 15 min at 100 C, and cooled in tap \;ater; ahsorbancy was read in a colorimeter (Klett Co.; filter 6o) against a standard calibration curve prepared from glucose or fructose. The mixture was usually shaken once during the heating step to remove bubbles. Fructose was assayed as follows (10). To 2.0 ml of the sample were added 0.2 ml of 1-5^ cysteine, 4.0 ml of 95?i H2S0j^, v/ith cooling in an ice bath, and 0.2 ml of O.15J carbazole in absolute alcohol. The mixture vTas shaken, heated at 60 C for 30 min, and cooled in tap water; absorbancy was read in a colorimeter (Klett C.; .: filter 56), or in a Beckman EU spectrophotometer at 1^70, 560, 65O, and 750 mu (10) against a standard calibration curve prepared from fructose. Pentose \ia.s determined by the following modification of the orcinol method (lO). T-. 3.0 ml of the sample vrere added 3.0 ml of 0.15i FeCl^ in concentrated HCl (vlth cooling in an ice bath), followed by 3 drops of orcinol reagent (50O mg/ml of absolute alcohol). The mixture was shalien and heated for 10 to 20 min at 100 C. Absorbance was read in a photoelectric colorineter (Klett Co.; filter 64) against standard curves prepared from arabinose, ribose, or R5P. Assay for pqycerates. Glycerates were analyzed as follows (lU). The sample, in a volume of 0.2 ml, was added to 5.8 ml of 0.01^ 4,5dihydroxy-2,7-naphthalene-disulfonic acid (chromotropic acid). The mixt^ore was shaken and then heated for 30 min at 100 C, cooled, and the absorbancy read in a colorimeter (Klett Co.; filter 69) or Beckman

PAGE 29

22 DU spectrophotometer at 69O mi against a knovn standard solution. I^zymatlc assays for phosphorus compounds . An enzyme, or a mixture of enzymes, vae used to assay G6P, f6P, and UlffG. In each case, the enzyme activity was tested against knovn substrate preparations. The change in optical density uas measured by a Beckman model DU spectrophotometer using a quartz cuvette having a 1-cm ll^t path. The level of substrate in a sanqple was con^juted from the molar absorbancy value of the reduced pyridine nucleotides at 3^0 mu (6.22 X 10 ), and this valvje vas multiplied by the appropzlate factor to give the results reported herein. A method for the determination of millimicromolar amounts of ATP is described in more detail. G6P and P6P . g6P was assayed by following the reduction of TPH in the presence of gluco8e-6-phosphate dehydrogenase (g6PD) ( 37) • G6j?D G6P*TPB ^ 6-phosphogluconate-HPPHH+H*' The amount of g6p vas calculated from the increeise in OD^i^q according to the expression ( 0D)(3.0) G6P (umoles) = 6.22 A typical reacticai mixtiire contained 1 .0 ml of 0 .04 M glycyl glycine buffer (pH 7-5), 1.0 ml of 0.02 M MgClg, 0.2 ml of TPK (4 rag/wl), 5 ul of g6PD (Sigma Type V), and G6P, in a total volume of 3.0 ml. P6P w3£ assayed in the same -my in the presence of G6PD and phosphohexose isomerase (50). PHI PfiP — > g6p Uridine d iphosiAate glucose (UlffG) . WOPG was measured by

PAGE 30

23 following the reduction of DPN in the presence of uridine diphosphate glucose dehydrogenase (UlffGD), \ftiich is specific for the reaction UDPGD unP(j+2ISPN ^ UII»-glucuronate+2DPHa UWGD was prepared from, calf liver, according to Maxwell et al. (56) The amount of UlffG was calculated from the increase in OD^j^q according to the expression ( OD)(3-0) UlffG (uncles) = 12.0 A typical reaction mixture contained O.06 ml of DPH (35 rag/ml, O.3 ml of 1 M glycine huffer, pH 8.7, 0.5 ml of UDPCH), the saarple containing UDPG, and water to a final vol of 3.0 ml. Microdetermi nation of ATP with firefly lantern extract . The intensity of the luminescence is proportional to the ATP concentration (W). As little as 2 X lO"^ umoles of ATP can he measured with a Farrand i^otofluorometer, using an extract prepared from firefly lantems . The extracts were prepared by the following modification of the method of McElroy (U8 ) . Fifty mg of dehydrated firefly lantems (Sigma Chemical Co.) were ground in a small glass homogenizer (Kontes Glass Co.) for 3 min in 1-1.5 ml of 0.1 M NagAsOi^ huffer, iffl 7.4, at 1-k C. The extract was transferred to a gradxiated test tube. The homogenizer was rinsed twice with cold buffer, the rinsings and extract were combined, and the volume was made up to 5.0 ml with buffer. Cell debris was sedimented by centrifugation and discarded. Fifty mg of MgSOj,. 7^ were dissolved in the supernatant liquid. This

PAGE 31

2k preparation is herein referred to as firefly lantern extract (FEE). All manipulations were done in the cold. Filtration of the hotnogenate (71) was not els convenient as centrifu^tion, and activity of the filtered preparation ms ahout lower. The FFE retained 90^ of full activity for 1 week at 5 C. A typical reaction mixture contained 0.2 ml of FFE, the sample to be assayed, and water in a total volume of 0.8 ml. Each preparation was calibrated a.gSLinst known ATP samples. The galvanometer was read 30 sec ai^er adding the sample. Full scale deflection (lO.O units) was usually produced by 0.2 rniimoles of ATP. The presence of transphosjdiorylases in the FFE was confirmed by several experiments in which GTP or GTP plus AEP were added instead of ATP (11). Trichloroacetic acid extracts of com root tips were assayed in the following manner. A portion of the extract was diluted with water to a Bxii table concentration, and a 0.2 ml sample was assayed as decribed above. The galvanometer reading was multiplied by an appropriate factor to determine the total ATP in a known weight of plant tissue . Single root tips were assayed as follows. A wei^d, 2.0 cm root tip (25-30 mg) was homogenized in 1.0-1. 5 ml of I.5 M perchloric acid for 30 sec at 1-k C. Tiuz homogenate was transferred to a small centrifuge tube, and the homogenizer was rinsed three times with about 0.5 jol of cold, water. The pH was adjusted to 7.2 with KOH (I.5 M and 0.I5 M), and the extract was centrifuged. The supernatant fluid was transferred to a graduated test tube; the precipitate was suspended in cold vater and centrifuged. The supernatant fractions

PAGE 32

25 were combined, and made up to 5.O ml with water. All monlpiilations were done in the cold. A 0.1 to 2-ml portion of this extract was assayed with FFB. Analysis ftrom UV absorbancy measurements . Absorbency was measured at 260 and 290 mu on the fractions eluted from Dowex 1 columns. Where ever the ratio 260/290 was high, the presence of a nucleotide was suspected, and measurements at additional wavelengths were taken to characterize the nucleotide (61).

PAGE 33

RESULTS Powex l-Chlorlde Chronatography of Kncrwn Coag>o\mds Test solutions containing mixtvireG of knovm P compounds were ckromatographed with Dowx l-chloride columns. The elution sequences were similar to those previously reported (1-3): AMP, OSP , f6F, and IP vrere eluted by 0.01 H HCl; ADP was eluted "by 0.02 K HClj FOP was eluted by 0.1 N NHi^Cl; ATP was eluted by 0.5 N Mi^Cl. The positions of the compounds were determined from chromatograms drawn from TP assay and OD250 measurements on each fraction. Another Dowex l-chloride colunn was elated with the five solvent systems. Each fraction of the "blank run" gave a zero response to TP assay and had negligible absorbance at 260 mu. The anthrone and cysteine -carbazole assay methods were applied to the P-containing fractions . Soias of the fractions showed surprisingly high absorbancy readings, due to the formation of bubbles. This result was most severe in the fractions containing the highest concentrations of ammonium ion, but could be minimized by thorough shaking of the test tubes \/ith a mechanical test tube shaker several minutes before absorbancy measurements were made . Dowex l-Formte Chro— itography of Known Coasfo\wd» A "blank run," 'osing 0 to 1 H formic acid, showed each fraction to be negative for TP. Absorbancy increased greatly with formate concentration in the 200-240 mu range, but was negligible in the near 26

PAGE 34

27 UV range. Anthrone and cysteine -carbazole assays on the same fractions gave low but erratic readings due to bubble formation in the presence of formate ion; vigorous shaking \»as necessary to minimize errors from this source. A test solution containing AMP, GIP, g6p, f6p, R5P, and IP ijas chromatographed . The anthrone, cysteine -carbazole, and orcinol assay methods were applied to each fraction; TP was also assayed on each fraction. AliP was measured from ODggQ readings. The compounds were eluted in the above sequence; AMP and IP were eluted separately, but the sugar phosphates overlapped. When using 10 min heating time with the orcinol losthod, and reading immediately, GIP, g6P, and F6P all gave virtually umneasurable readings (less than 1^ of the readings given by R5P) . Using a 20 min heating time increased the sensitivity by 3^, but increased interference from hexose phosphates by 10^. The color was stable; readings after 2k hr at room temperature indicated intermediate sensitivity and specificity. The assay appears to be extremely specific for pentose in the presence of large amounts of hexose. Cysteine-carbazole method was not specific for fructose phosphates, as reported by Bartlett (13), but absorbancy readings for GIP and g6p were about 355& as high as for an equivalent amount of P6p. Only a trace of reaction with cysteine -cajrbazole was given by R5P. Ar other test solution, containing ULPG, FDP, 2PGA, and 3PGA, vas chromatograi*ied with a Dowex 1-formate column, \rtiich was eluted with 0 to H formate buffer, containing four parts of formic acid and one part of amnonlum formate (pH 3-0). UDPG and the two glycerates were eluted simultaneously.

PAGE 35

28 Dovrex 1-Clilorlde Chr-' Taatography of Corn Root Tip Extracts Table 1 shows the levels of TP and IP in pooled trichloroacetic acid extract of 63.2 g of root tips, before and aifter passage through a Dowex 50 column; recovery of IP was nearly quantitative, while 15^ of the TP was retained on the cation exchange resin. Table 2 shows TP and IP levels in several Dowex 50-ti«ated root extracts. Pigs. 1-i; show the res-alts of Dowex 1-chloride chromatography of root tip extracts. The conposition of the eluates could not be determined from these data. It appeared from these results that (a) Qnly a crude separation wac obtained by the Dowex 1-chloride column (b) the compounds of interest were masked by the presence of various materials giving color formation \rLth TP and sugar phosphate analyses, and others having absorption in the UV spectrum (c) formation of bubbles obscured results in the three sugajr assays. Dowex l-Foroate Chromatography of Eluates from Dowex 1-chloride Separations of Com R^ot Tip r::rbract3 Since Dowex 1-chloride chromatography apparently did not seixirate sufficiently the phosphorus compounds of interest, and since considerable amounts of unknown materials appeared to mask the results, the solutions eluted from Dovrex 1-chloride columns were chromatographed again on Dowex 1-f ornate columns: each of the five pooled eluates from the chloride columns was passed through a Dowex 1-forraate column, and the anions were eluted either with formic acid or a formic acidammonium formate buffer. 0.01 N HCl section . Fig. 5 shows the results obtained from the rechromatograiiiy of the 0.01 K HCl eluate from a trichloroacetic acid extract of I8.6 g of root tips, using 0 to 1 N formic acid for elution. Similar res\ats were obtained from extracts of I9 to 71 g of root tips.

PAGE 36

29 TABLE 1 PHDSPffi)HlJS COMTENT OF A TRICHLOROACETIC ACID EXTRACT OF 63 .2 GRA^B OF CORK ROOT TIPS, BEFORE AND AFTER TREATMEMT WITH DOWEX 50 P, ufflolea/g 5b Recovery IP TP IP TP Before Dowex 50 3.09 6.6V After DowBx 50 3.03 5.63 98 85

PAGE 37

30 TABLE 2 PHDSPHDRUS COHCOT? OF SEVERAL TRICHLOBOACEPIC ACID EXTRACTS OF CORK ROOT TIPS, AFTER TREATMan? WTIH DOWEX 50 Wt of root, g IP, ujnoles/g TP, UBioles/g 57.1 7.50 7k. h 3.03 3.2K 3.03 5.63 29. e 2.88 7.10 Av. 3.2k Av. 6.37

PAGE 38

31 LITERS THROUGH COLUMN LITERS THROUGH COLUMN Fig. 1. TP Fig. 2. OD260 O OS .1 .1 .'4 .4 J .i .i .4 ' .i io a iJi 1:3 •01 .Oa .IN .1H .5N NHCI NHCI NH4CI NH4CI NH4CI LITERS THROUGH COLUMN 1 .01N HCI .02NHCI .IN .2N .SN NH4CI NH4CI NH^Ct LITERS THROUGH COLUMN Pig. 3. Anthrone (hexose) Fig. k. Cysteine-carbazole (kfitose ) Pigs . 1-1* . DOWEX l-CHLORIDE CHHOMATOORAm OF TRICHLOROACETIC ACID EXTRACTS OF CORN ROOT TIPS . The Dowex 50-treated extracts were run through Dowex 1chloride columns, and the anions were desorbed by elution with the indicated solutions.

PAGE 39

32 LITERS THROUGH COLUMN Fig-are 5 . DO\ffiX l-PORMATE CHROMATOGRAPHY OF THE VIEAK ACID PBOSPHATE COMPOUHDS FROM A TKECHLOBOACETIC ACID EXTRACT OF l8 .6 GRAMS OF CORK ROOT TIPS. The pooled, neutralized, and diluted 0.01 K HCl section of a Dowex 1chloride sei>aration -was run throtigh a Dowex 1-formate column and the adsorbed anicms were eluted vith 1.3 1 of concave gradient 0 to 1 II fonalc acid. Assay methods: > TP: , Anthrone: , Orcinol.

PAGE 40

33 The chromatogram vas plotted from TP, anthrone, cysteine-carbazole, and orcinol values . The color formed irith the acthrone and cysteine car"bazole reagents was compared with the color fonned \ri.th fructose standard solutions, and was measured with the Klett colorimeter. The TP vas recovered in two well-defined peaks. Table 3 shows the recovery of TP from four root extracts. Linear and concave gradient elutions proved to be equally satisfactory; the only apx)arent difference was an overall shift of the position of the elution peaks. A large aiaount of absorbance in the anthrone method appeared in the first 1^00-500 ml of eluate. This was probably due to the presence of non-phosphorylated carbohydrates, since no phosphorus was detected in these fractions. The cysteine -carbazole and anthrone assay methods gave similar results \Aien applied to the fractions of Peak 1; addition of the values obtained with those fractions gave 1.46 umoles of ketohexose/g of roots \riLth anthrone and l.h^ umoles of hexose/g of roots with cysteine -carbazole. The fractions in Peak 2 were essentially negative with anthrone, except for a lov "background" absorbance probably due to the persistent elution of non-phosphorylated carbohydrates. The orcinol method of pentose assay, measured against a R5P stock solution, gave a total of O.387 umoles of pentose/g of roots, with the peak values at the same elution position as Peak 1. The orcinol result was about \Aiat woxild be expected from the anoiait of hexose monophosphate present, as calculated from anthrone or cysteine -carbazole methods. There is, then, no evidence for the presence of measurable amounts of R5P. Assay for IP was negative for the fractions in Peak 1; IP assay on the fractions in Peak 2 gave the same values as TP assays (Table 3), indicating that Peak 2 consists of IP.

PAGE 41

3h

PAGE 42

35 Only the first 200-300 ml of eluate absorbed very significantly at 260 ma; no phosphorus was detected in those fractions. The Peak 1 fractions from on extract of 22.7 g of root tips were tested for ketose by the cysteine -carbazole method using the Beckman DU spectrophotcaneter, with the following results. Readings vere taken on each fraction at l»-70, 560, 65O, aad 750 mi (lO). Optical density readings were some^Aat lower at 650 than at 1^70 mu, indicating no triose idiosphate was present in the fractions tested. There was negligible absorption at 750 mu. Based on the value 0.6 for 0.1 umole of ketohexose (l-cm li^t path), the ketohexose in Peak 1 was computed to be 0.39 umolee/g. This accounted for h^^ of the TP in the Peak 1 fractions. The proced^ore was repeated with the fractions from another TOot extract (71.1 g); kO^ of the TP was accounted for as ketohexose based on cysteine -carbaaol£ assay. Since the cysteine -carbazole method was found in preliminary eacperiments to suffer from rather high levels (35^j) of interference from GIP and g6P, the absolute values obtained in a mixture did not seem significant in themselves. However, inspection of the ratios of cysteine-carbazole readings/TP content computed for each individual fraction showed the highest values in those fractions corresponding to the descending slope of Peak 1. This is the elution position where p6p has been reported to occur (13), and which vas confirmed in these studies. The pooled fractions of Peak 1 were analyzed enzymatically for (S6P; the results indicated the presence of O.58 umoles of G6P/g of root tips, accounting for 56^ of the TP in the peak. An extract of 38.3 g of roots was tested for GIP by measuring acid-labile (7-mln) P in the ftractions in Peak 1 (Fig. 6). All

PAGE 43

36 to ill l."2 LITERS THROUGH COLUMN Figure 6. DO'.ffiX 1 -FORMATE CSBDimOGBAFSI OF THE SUGAR MONOPHOSPHATES (PEAK 1) FROl^ m EXTRACT OF 3^-3 GRAMS OF CORK ROOT TIPS. (See Fig. 5 )• Linear gradient. Assay laethods: , TP; , acid-labile (7 min) P.

PAGE 44

37 fractions were negative for IP; i.e., before hydrolysis. Determination of 7-min P on the same fractions gave peak values in the region of the ascending slope of Peak 1. This is the elution position previously reported for GIP (iS), and confirmed in these studies. Ho other phosphorus concpound is kno-vm to be eluted in this region, and none of the other sugar phosphates is appreciably labile after 7 inin in 1 N HCl at 100 C. Total GIP was computed to be O.O5 unoles/g of root tips. 0.02 N HCl section . Fig. 7 shows the restilts obtained from the rechromatography of the 0 .02 K HCl eluate from a trichloroacetic acid extract of 57.1 g of root tips, using 0 to 1 H aamonlum formate for elution. Recovery of P \iaB 60^, about 90^ of ^ch was in the two peaks having maximum values at h^O and 720 ml. ODgj-Q and OD^^^q was measiired on each fraction. Infinitely large readings were obtained in the first 3OO ml at both wavelengths. The UV spectra of these fractions did not resemble those of the nucleotides, and very low levels of P were foxjnd in those fractions. Another tall UV peak having the same characteristics, occurred at 77O-01O ml. Three fractions, at h80, k90, and 5OO ml, (Fig. 7) were assayed for glycerate by the chromotropic acid method {ih) . In each case, the spectrum was the same as the characteristic spectrum produced by known MPG under the same test conditions. Further, the ratio of glycerate to TP was about 1:1 for each of the three fractions. From this evidence it was concluded that the three fractions contained essentially pvire MPG; the concentration was calculated to be 0.01 umoles of MPG/g of root tips. The UV spectra of the fractions collected at 720 to 750 ml

PAGE 45

38 S .20 Ul o.15 O .10 05 T — r 1 r T r LITERS THROUGH COLUMN Figure ?• DOV/EX 1 -FORMATE CHROMATOGRAPHY OF THE 0.02 N HCl SECTION OF A TRICHLOROACETIC ACID EXTRACT OF 57.1 GRA^B OF CORK HOOT TIPS. The pooled, neutralized, and diluted 0.02 ^ HCl secticm of a Dowex 1-cliloridfi separation was run through a Dowex l-formate column, and the adsorbed anions were eluted with 1.2 1 of linear gradient 0 to 1 H ammonium formate buffer. Assay: TP.

PAGE 46

39 resembled that of the adenosine phosphates. The absorboncy ratio 250/260 averaged O.78 for these fractions and the ratio 280/26O averaged O.lC. The pH of the eluates \ra.s G.k. These ^/alues vere identical to those previously reported for AEP at pH 7.0 (see Table 4). The nucleotide content of the saoe four fractions yac calculated from the molar absorbancy value of the adenosine phosphates, 1^ .h X 10"^ (61). The i-atio of TP to adenine (oD^^q) of the saias fractions i/as 2:1. A Dowex 1-forraate chrcoatogram of an authentic sanple of AUP had the peeik at the saoe elution position. From this evidence it yes concluded that the compound was ADP; the ADP level was calcxilated to be 0.85 umcles/g of root tips. 0.1 U lIHi^Cl, 0.2 K IJHi^Cl, and O.5 1! Mj^Cl sections. These three eluates from Do-^rex 1-chloride chromatography were each chrooatographed with a formate buffer, using the same techniques described above . In neither instance was there an appreciable recovery of most probably due to the presence of relatively hi£^ concentrations of chloride ion. Direct Doi/ex LFormte Chromatography of Com Root Tip Extracts Root tip extracts, each representing 50-75 g of tissue, were run through Dowex 1-formate columns aTter purification irlth Dowex 50 resin. The adsorbed compounds were eluted with 0 to It K aumonium formate buffer, pH 3.0, containing four parts of formic acid and one part of aranonium formate. Linear and concave elution gradients were used. The chromatograras were monitored, as usual, by TP assay on each fraction. Comparison of a concave gradient chromatogram (Fig. 8) \/ith a linear gradient chromatogram (Fig. 10) sho\jed the same sequence of major peaks; but the concave gradient gave better separation of the weakly acidic compounds.

PAGE 48

iw sad saiow

PAGE 49

1*2

PAGE 50

Iv3 1.1 1.0 .9 .8 ui .6 O S .5 .4 I I I r— — I— r T 1 1 1 I 12 .1 .i .3 .4 .i .6 lo 1.1 i3 iA i.s l6 LITERS THROUGH COLUMN Fig'ore 10. DIRECT DOVffiX 1-PORMATE CHRCMmXIRAPHY OF A TBICHLOROACETIC ACID EXTRACT OF 71.9 GRAMS OF COHN ROOT TIPS. The procedure folloved was deecribed in Fig. 8; linear gradient elation. Assay: TP.

PAGE 51

In a typical experiment (Fig. 8), using 5I.8 g of root tips, 6-75 tanoles of P/g of root tips were added to the column, and 5 '52 vuaoles (82^) vere recovered in the individual fractions. Of the P recovered, from a^/erages of several runs, 1^-92 umoles (87y&) appeared in the four largest peaks, as follo\re: Peak 3, O.93 umoles/g; Peak k, 3.61 umoles/g; Peak 7, 0.10 uiaoles/g; Peak 10, 0.25 umoles/g. Peak 3 corresponded to the hexose monophosphate peak (Peak l) in Dowex 1-formate chromatography of the 0.01 H HCl Dovex 1-chloride eluate (Table 3). Peak k was IP, corresponding to Peak 2 in the same table. The fractions of Peak 3 were pooled, freed of ammonium and formate ions, and analyzed enzymatically for f6P. Analysis shoved the presence of 0.15 umoles of F6p/g of root tips: this accounted for lh<^ of the TP in Peak 3Fig. 9 shows OD25Q sad. ODg^ measurements of the eluates from concave gradient chromatography of 51. 8 g of root tips. Extremely hi^ levels of absorbancy at both wavelengths were found in the first 300 ml, indicating the presence of large amounts of unknown materials absorbing in the near UV spectrum. A UV peak, possibly representing a nucleotide, was found at 39014-50 ml. The position of the peak did not correspond exactly to Peak k (Fig. 8). Using the molar absorbancy value 15.4 X 10 , the total UV-absorbing material would be less than 0.02 xaaole/g, or 0.7^ of the phosphorus in Peak U, on a monoidiosphate basis. The identity of the canpound was not determined. A small UV-absorbing peak was found at 950-1000 ml; it was not identified. Another small peak at 1380-lUlvO ml was not identified. A fairly large peak at 160O-1700 ml did not correspond to the TP

PAGE 52

h3 chromatogram, and its identity was not ascertained. Two peaks were found at IO3O-II3O ml and n^O-lSkO ml. The fractions making up each peak in Dowex 1-formate direct chromatography were pooled, anmonium and formate ions were removed, and UV absorbancy was measured on each combined peak (Fig. 11 ). Only Peak 6/T (combined) showed a UV absorption spectrum similar to a nucleotide. Peak 6 contained 4^ of the TP in the combined peak 6/7. The OD,^^ of Peak 6/T was 262 mu, and the complete UV absorption spectra at pH 2, T, and 11 (not shown) were similar to spectra of the uridine nucleotide, and unlike the spectra of the other nucleotides. The absorbancy ratios 250/260 and 280/260, given in Table k, indicated that Peak 6/T probablycontained UTP or UDPG, or both. Enzymatic assay of the peak indicated the presence of O.13 umoles of UDPG/g of root tips, corresponding to 0.26 'jmoles p/g root tips; 38^& of the TP in Peak 6/7 can be accounted for as UDPG. Orcinol, anthrone, chromotropic acid, cysteine-carbazole , and acidlabile (7-min) P assay methods were aiiplied to each of the pooled peaks. Ho clearcut res^olts were obtained, because the peaks all contained impurities >Aiich interfered with colorimetric assays . ATP Assay of Com Root Tip Extracts The firefly lantern extract was used to measure the ATP level in trichloroacetic acid extracts of root tips. The results indicated 0.15 uiaoles of ATP/g of root tips in the acidfree extract. When the extract, containing trichloroacetic acid, was analyzed after neutralization with 0 .1 M Tris buffer to pH 7 , the apparent ATP concentration was only 20^ of that observed in the acid-free extract, possibly due to a salt effect. ATP content of a perchloric acid extract of a single root tip was O.18 umoles/g.

PAGE 53

Figure 11. UlffR/WIOLETP SPECTRA. OF THE POOLED TP PEAKS FROM DIRECT DOVIEX 1 -FORMATE CHROMATOGRAPHY OF TRICHLOROACETIC ACID EXTRACTS OF CORII ROOTS. The fractions of each nijnbered peak (see Figs. 3 and 10) were pooled, acnaonium ion was removed with Dowex 50 (hydrogen form), formic acid was removed by extracting with ether; each solution was neutralized before its UV absorhancy was measured.

PAGE 54

47 Phosphorus CcagpouDdB Identified In the Root of the Etiolated Com Table ^ shovs tbe concentratlozis of the F coa^unds foimd In the seedling root . The values for MPG and. MP are, uodoubrtedly, iBuch too lov, because about kO^ of tbe TP failed, to adsorb to tbe Dovex 1-formte colxaon.

PAGE 55

HQ TABI£ 5 PHOSPHATE CCHffCXniDS OP CORK ROOT TIPS Con5)ound uiaales/g of corn root tips 2.65-3-86 GIP .05 .58 p6p •15 UEPG .13 MS .85 ATP .15 MPG .01 Range of values from four extracts. Each other value from a single extract.

PAGE 56

Discussion Previous workers have encountered difficulties in the separation and analysis of the phosphoras compounds in plant tissues, and one of these, Albaum ( 3 ), declared the use of ion exchange chromatography for this purpose "not feasible," because of the syamping of the analyses at e^nery step by large quantities of phosphorus compounds of various compositions. Considerable difficulty vas also encountered in these studies for similar reasons. The com seedling root tissue contained high levels of phosphorus compounds of unkno^ai composition, i^ch Mere difficult to separate from the glycolytic intermediates and nucleotides which were of primary interest. However, the results of the investigations reported in this paper indicate that anion exchange methods show promise for the separation and quantitative analysis of those conpo-onds of com seedling roots, and it is belie^yed that the foi-ther application of these methods ;/ill result in the analysis of many others. It was concluded that the use of colorimetric methods for the assay of sugar phosphates in mixtures is of United value in such work. An exception was the orcinol assay method for pentose. The phosphor-as compounds were not separated sufficiently on Dowex 1chloride columns to analyze them quantitatively from TP assay, UV absorbancy meas-arements, and colorimetric sugar analyses on the k9

PAGE 57

50 eluted fractions. The eluates from Dovex 1-chloride columns could be rechroiaatographed on finer mesh Dowex 1-fonnate columns, however, resulting in further separation of phosj^orus compounds . The compounds thus separated appeared to be in almost pvire form, except for the hexose monophosphates, •vrfilch were not separated from each other under the test conditions. This method of re chromatography of chloridecontaining eluates «as unfortunately limited in its usefulness to eluates 0.02 H or less in chloride ion, because of the swamping of the phosphorus compounds at hi^ chloride concentrations. Slower elution of the Dowex 1-chloride column with smaller amounts of chloride eluent may overcome this difficulty. The use of Dowex 1-formate columns to separate directly the tissue extracts, without prior separation on Dowex 1-chloride offers promise. The ammonium and formate ions, unlike the chloride ion, can be essentially removed from the eluates. Dowex 1-formate rechromatography of selected portions of these eluates, using various ansnonium formate buffers, would be exacted, to provide further separation . The enzymatic analyses proved to be useful vben dealing with eluates of varying degree of purity. It may prove practical, as well as expedient, to perform such analyses on the five relatively mixed eluates from Dowex 1-chloride columns . The firefly lantern extract (lucif erase) method was found to be an extremely sensitive method for measuring ATP in plant tiss\ie. The concentrations of the phosphorus compounds identified in acid extracts of com roots should be confirmed by additional tests.

PAGE 58

Methods were described for the separation and analysis of glycolytic intermediates in etiolated com seedling roots, with the use of ion exchange col'jmn chromatograiiiy . The following compounds vrere identified: inorganic phosphate, glucose -1 -phosphate, glucose6-phosphate, fructose-6-phosphate, monophosijhoglyceric acid, adenosine diphosphate, adenosine triphosphate, and uridine diphosphate glucose . 51

PAGE 59

1. Abood, L. and Goldaan^ E. I&hlMtlon of phosptaozylatlaa durliic electrical excitation of frog nerves. Am. J. Physiol. l8i>:329. 1956. 2. Aisenberg, A. C . Sugnr phosphate levels In the mitochondrial Pasteur effect. J. Biol. Chen. 23k'Ml. I959. 3. Albman, H. 0. ^le Isolation of adenosine triphosphate from plant tlssite. Arch. Blochem. 27:130. 1950. k. A l h a i na, H. 6. The aetabollsm of phosphorylated coopounds In plsats. Ana. Bev. Plant Physiol. 3:351952. 5. Albaum, H. 0. and Ogur, M. An adenlzie -pentose-pyrophosphate from plant tissues. Arch. Biochen. 15:158. 19JI7. 6. Alhaum, H. 6., Schats, A., Hutner, S., and Hlrshfeld, A. Phosphorylated coiqx>unds In Eviglena. Blochem. J. 29:210. 1958. 7Albaun, H. G., and Unibreit, W. W. Differentiation between riho8e-3-pho8phate8 and rihose5 -phosphate by laeaas of the orcinol-pentose reaction. J. Biol. Chen. 167:369. 19^7. 8. Albaum, H. 6., and Iftabreit, W. W. Phosphorus transformtions during the development of the oat enhryo. An. J. Botany. 30:5^2. 19^*3. 9. Aim, R. S., WlUians, R. J., and Tiselius, A. Qiadient elutic» analysis. I. A general treatment. Acta Chen. Scand. 6:826. 1952. 10. Ashwell, G. Colorlmetric analysis of sugars, p. 73. In S. Colowiclt and II . Kaplan, (eds.). Methods In enxymDlogy.^III. Academic Press Inc., Mev Tork. 1957. 11. Balfour, W. M., and Samson, P. E. Transpho»i*ioiylaaes In the firefly lantern. Arch. Blochem. Biojhys. 81»:ll»0. I959. 12. Bartlett, G. R. Human red cell glycolytic intenoedlates . J. Biol. Chem. 23l|-:l*49. 1959. 13. Bartlett, G. H. Methods for the Isolation of glycolytic intermediates by colunn chrooatography with ion exchange resins. J. Biol. Chem. 23l^:1^59. I959. 52

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53 ik. Bartlett, G. R. Colorimetric assay methods for free and jAiosphorylated glyceric acids. J. ttLol. Chem. 23k:k69. 1959. 15. Bell, R. D and Doisy, E. A. Rapid colorimetric methods for the detexmLnatlon of fiiosphojrus in urine and hlood. J. Biol. Chem. 55:6?. 1920. 16. Benscoi, A. A , Bassham, J. A., Calvin, M., Goodale, T. C, Haas, V. A., and Stepka, W The path of carbon in photosynthesis. V. Paper chromatography and radioautography of the products. J Am. Chem. Soc 72:1710. 1950. 17. Berenhlum, I , and Chain, E. An iii?>rovecL method for the coloriBietric determination of phosphate. Bioche*. J. 32:295. 1938. 18. Berf^vist, R and Deutsch, A. Ion exchange chroioatography of nucleoside polyphosphates. Acta Chem. Scand. 8:1877* 195^ • 19. Bishop, C, Raakine, D. M., and T£LLbot, J. H. The nucleotides in normal human hlood. J. Biol. Chem. 23i*:1233. 1956. 20. Bock, R. M., and Tilng, Han-Sing. Devices for gradient elution in chrcmatography. Anal. Chem. 26:15^3. 195^. 21. Broioi, A. H. Determination of pentose in the presodce of large quantities of glucose. Arch. Biochem. 11:269. 19^6. 22. Cheng, Sze-Chuh. A sensitive method for ASS? and the determlnatlcoi of ASF, AnP and CrP in single nerve trunks. J. Hevuxtchem. 7:271. 1961. 23. Cohn, W. E. The anion-exchange separaticci of ribonucleotides. J. Am. Chem. Soc. 72:1^71. 195O. 2k. Cohn, W. E. and Carter, C E. Separation of adenosine polyphosphates by Ion exchange and paper chromatography. Federation Proc. 9:l6l. 195O. 25. Corl, G. T., Sleln, M., and Corl, C Crystalline d-glyceraldehyde-3-pho8^iate dehydrogenase from rabbit naiscle. J. Biol. Chem. 173:605. 1948. 26. Crane, R. K., and Lipoann, F. ^le effect of arsenate on aerobic phosphorylation. J. Biol. Chem. 201:235. 1953. 27. Diedrich, D. F , and Anderson, L. Gedactose l-phosphate in the intestinal tissue of the rat during galactose ahsorptlce. Blochim. Biophys. Acta ^5:^90. I96O. 28. Diedrich, D. F., and Anderson, L Separation of galactose 1idiosphate from other hexose phosphates by ion-exchange. AiulI. Biochem. 2:68. 1^1.

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51^ 29Dische, Z., Shettles, L. B., and Osnos, M. Hew specific color reactions of hexose and spectroidiotoinetric micro -Methods for Ujeir determination. Arch. Biocheia. 22:l69. 19^9* 30. Fiske, C. H. and SubhaRov, Y. The coloriaetric determination of jJiosphorus . J. Biol. Chem. 66:375* 1925* 31. GoBiori, G. A modification of the colorimetric phosphorus deteraination for use viUx the photoelectric colorimeter. J. Lab. Clin. Med. 27:95519^32. GoodBBn, M., Benson, A. A., and Calvin, M. Fractionation of phosphates from Scenedesaus "by anion exchange. J. Am. Chem. Soc. 77:1*257. 195533. Greenyard, P., Brink, P., and Colowick, S. P. Scane relationships between action potential, oiiygen consus^ion and coenzyme content in degenerating peripheral axons. J. Cell. Coop. Physiol. H:375. 195^34. Banes, C A., and Isheruood, F. A. Separation of the phosphoric esters on the filter paper chromatograms . Bature164:1107. 19'*9. 35. Belhert, J. R.» and Brovn, K. D. Factors influencing quantitative determ nation of methylpentoses and ketohexoses vith anthrone. Anal. Chem. 27:1791195536. Horecker, B. L., and ^oyxnlotls, P. The enzymatic production of ril}ose 5-P^8P^^e from 6-phDsphogluconate . Arch. Biochem. Biophys. 29:232. 1950. 37. Horecker, B. L., and Wood, WA. Glucose -6 -phosphate, p. I52. Ir Colovlck and B. Kaplan, (eds.). Methods in enzyaology. AcadeBdc Press Inc., Bev York. 1957* 38. Hurlbert, R. B., Shmltz, H., Brunm, A. F., and Potter, J. B. Bucleotlde metabolism. II. Chrcn&tographic separation of acid-soluble nucleotides. J. Biol. Ch^. 209:23. 1954. 39. Khym, J. X., and Cohn, W. E. The separation of su0ur phosphates hy ion ftxchnngp vith the uae of the borate con^lex. J. Am. Chem. Soc. 75:1153« 1953kO. Komherg, A. Beversihle enzymatic synthesis of diihosphopyridine nucleotide and inorganic pyrophosphate. J. Biol. Chem. 182:779. 1950. 41. Kunltz, M. Crystalline inorganic pyrophosphatase isolated frc« Baker's yeast. J. Gen. physiol. 35:423. I952. 42. lAAiyette, et ATP-creatlne treaxsphosphorylase, p. 605. La S. Colovlck and B. Kaplan, (eds.). Methods in enzymology. II. Academic Press Inc., Hev York. 1957.

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55 1^3* lekshmanan, T. K., and Lleberman, S. An Improved method of gradient elutlon chromatography and Its application to the Beparatlon of urinary ketosterolds . Arch. Blochem. Blophys. 53:258. 195^. Uk, Laughnan, B. C, and Martin, RP. Methods and equipment for the study of the incorporation of phosidiorus hy Intact harley plants in eatperlments of short duration. J. Exp. Botany. 8:272. k^. LePage, a. E. Methods for the analysis of phospborylated intermediates, p. 193* In Ihnbrelt, Burris, and Stauffer, (eds.), Manometric techniques and related methods for the study of tissue metabolism. Burgess Publishing Co., Minneapolis. 1951. U6. Lepage, 0. E., and Umbreit, W. W. niosphorylated carbohydrate esters in autotropic bacteria. J. Biol. Chem. ll*7:263. 191*2. kT. McElroy, W. D. The energy source for blolumnescence in an isolated system. Proc. Hat. Acad. Sol., U.S. 33:3^* 19^*7 • UQ. McElroy, •.D. Factors influencing the response of the bioluminescent reaction to adenosine triphosidiate . Arch. Blochem. 22:1*20. 1949. 1*9. McElroy, W. D. Properties of the reaction \itlliaing adenosinetriphosphate for blolumiinescence . J. Biol. Chem. 191:547. I95I. 50. Mandl, I., and Neuberg, C. Ketohexose phosphates, p. 162. In S. Colovick and N. Kaplan, (eds.), Methods in enzymology. III. Academic Press Inc., New York. 1957. 51. Markham, R., and Smith, J. D. Chromatographic studies of nucleic acids. I. A technique for the identification and estimation of purine and pyrlmldlne bases, nucleosides and related substances. Blochem. J. 45:294. 1949 . 52. Marre, 2., and Fortl, Q. Metabolic responses-to-a\ucLn. III. The effects of auxin on ATP level as related to the auxin induced respiration increase. Physiol. Plantarum 11:36. 1954. 53* Marre, B., Teuboer, F. 0., and Mumeek, A. E. Growth and phosphorus metabolism in tomato ovaries. I. Changes in phosphorus fractions. Am. J. Botany. 41:"^. 1954. 54. Marsh, B. B. The estimation of inorganic phosphate in the presence of adenosine triphosphate. Blochim. Biophys. Acta. 32:357. 1959. 55* Martonosi, A. Chromatographic separation of phosphate conqpounds. Blochem. Blophys. Res. Conmiun. 2:12. i960.

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56 56. Mawell, B. S. et Determliiatiaa of UIFG and OTP by nea&8 of UlffG dehydrogenase, p. 97^. ^ S. Colovlck and H. Kaplan, (ed£.)> Methods in e&zymology. HI. Academic Press Inc., Hew lark. 1957. 57. Mtlls, 0. C, Burger, D. 0., Schneider, M., and Levin, W. C. Icsx exchange analysis of organic phas;{^te conqpounds of erythrocytes from individuals with neoplastic diseases. J. Lab. Clin. Med. 58:725. 196l. 58. Minard, P. H., and Davis, R. V. The effects of electroshock on the acid-soluble phosphates of rat brain. J. Biol. Clien. 237:1283. 1962. 59. Borris, D. L. Quantitative detennlnatioa with Dreywood*s enthrone reagent. Scioice 107:254. 19^8. 60. Ochoa, S., Sedles, J.Q., and Ortis, P. J. Biosynthesis of dicarboxylic acids by carbon dioxide fixaticoi. J. Biol. Chem. 187:863. 1950. 61. Fabst Laboratories, Circular OR-7, Ultraviolet Absorption spectra of 5 '-ribonucleotides, March 1955* 62. Pontls, H. 0., and Blunson, B. L. A method for ths sepaaration of nucleotides by concave gz«dient elution. Blochim. Biophys. Acta 27:618. 1958. 63. Backer, £. Spectropbotonaetrlc BeasureBent of hezokinase and phospbohexbkinase activity. J. Biol. Chem. l6j:8k3. 19^7. 6h, Holnftdns, P. W., and LipBoan, P. Separation of the tvo enzymatic phases in active sulfate STntheais. J. Biol. Chem. 233:681. 1958. 65. Boe, J. H. The detendmation of sugar in blood and spinal fl\iid with anthrooe reagent. J. Biol. Chem. 212:335. I955. 66. Rowan, K. S. fbosphorylated compounds in planta. I. Adenosine and uridine 5 'phosphates in pea seedlings. J. Eaq>. Botany. 8:256. 1957. 67. Rowan, K. S. Phosphorylated cos^tounds in plants. II. The estimtioo of hexoae phosphates and adenosine pyrophosphates in plant tissue by the method of Slater. J. ficp. ^tany 9:J^37. I958. 68. Rowan, K. S., Saamon, D. S., and Tuner, J. S. Phoaphorylation as a possible factor in the Pasteur effect in plants. Bature m:3331956. 69. Seraydarian, K., Moimnaerts, W. P., and Wallner, A. Etozymtic fluorcaaetrlc methods for the raicrodetermination of hexose l4iosphates in muscle. J. Biol. Chem. 235:2191. 1960.

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57 70. Slater, E. C Spectrophotooetrlc detexnlnatloa of fructose 1 :6-dlpbosphate ^ hexoseooaophosphates, adenoslnetrlpliosptaate ezkd adenosine dlpbosfbate . Blochem. J. 53:1^7' 1953 • 71* Strehler, B. and Totter, J. BFirefly lumlnesceuce In the study of energy transfer mecharlsias . I. Substrate and enxyoe determination. Arch. Blocboa. Blo|il]ys. kO:2B. 1952. 72. Wade, H. E. Fractlcaiatlcaei of phospbate esters on Ion eTrhange resin by a nev system of pH gradient elutlon. Blochem. J. 77:53'>i960. 73 • Veiner, H. The content of adenine nucleotides and creatine phosphate In brain of normal and anaesthetized rats: a critical study of some factors influencing their assay. J. Heurochem. 7:2la. 1961. Tk. MyaAt, G. R. The purine and pyrlmidlne ccaqposltion of deoxypentose nucleic acids. Blochem. J. k6i^. 19^.

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SKXSAFEICAL SSSECS. Btyron H. Wise this born 12 February 1925 In Galaesvillfi, Florlde . Ha gradiiated from P . K . Ycao^ Laboratory School In 19^. Os attended the University of Florida from 19^2 to 19kk. After service in the Arc^, he re-entered the University in 1914-6, and received the Degree of Bachelor of Science, vlth Honors, in 191^9. His major svihjects were Biology, Chemistry, and Psychology. He received the Degree of Macter of Science in Agrictilture in 1953 from the University of Florida. His major subject \BS Botany, specializing in Taxonony. He took tvo years of graduate vork. at Washington University^ St. Louis, KtLssoiirl. For fo\ir years he was an industrial research chemist. In 1959 ^ started a graduate program in the Oepartioent of Botany, University of Florida, majoring in Plant Physiology. He is a candidate for the Degree of Doctor of Philosophy, to be auarded in December, 19^2. His is a member of the folloving honorary societies: Phi Eta Sigma, GeitBa Sigma Epsilon, Phi Sigma, and Fhi Kappa Phi. He is married to the former Winnie Sue Moss. 58

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This dissertation was prepeur^d under the direction of the chairman of the candidate's supervisory conanittee and has been approved by members of that committee . It was submitted to the Dean of the Colleee of Agriculture and to the Graduate Council, and was approved as partial fulfillment of tlie requirements for the degree of Doctor of Philosophy. December, I962 Dean, Graduate School Supervisory Ccamittee: