Title: Experiments in marine biochemistry: I. Homarine metabolism. II. Chemoreception in Nassarius obsoletus
CITATION PDF VIEWER THUMBNAILS PAGE IMAGE ZOOMABLE
Full Citation
STANDARD VIEW MARC VIEW
Permanent Link: http://ufdc.ufl.edu/UF00098336/00001
 Material Information
Title: Experiments in marine biochemistry: I. Homarine metabolism. II. Chemoreception in Nassarius obsoletus
Physical Description: vii, 75 leaves. : illus. ; 28 cm.
Language: English
Creator: Hall, Elizabeth Ruth, 1947-
Publication Date: 1974
Copyright Date: 1974
 Subjects
Subject: Biochemistry -- Research   ( lcsh )
Marine biology   ( lcsh )
Shrimps   ( lcsh )
Biochemistry and Molecular Biology thesis Ph. D
Dissertations, Academic -- Biochemistry and Molecular Biology -- UF
Genre: bibliography   ( marcgt )
non-fiction   ( marcgt )
 Notes
Thesis: Thesis--University of Florida.
Bibliography: Bibliography: leaves 72-74.
General Note: Typescript.
General Note: Vita.
 Record Information
Bibliographic ID: UF00098336
Volume ID: VID00001
Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
Resource Identifier: alephbibnum - 000585097
oclc - 14181267
notis - ADB3729

Downloads

This item has the following downloads:

experimentsinmar00hall ( PDF )


Full Text









EXPERIMENTS IN MARINE BIOCHEMISTRY:
I. HOMARINE METABOLISM
II. CHEMORECEPTION IN Nassarius obsoletus










By

ELIZABETH RUTH HALL


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


1974













ACKNOWLEDGEMENTS


The author would like to thank Dr. Samuel Gurin whose

guidance, support, and tolerance proved invaluable through-

out her graduate career. Furthermore, she would like to

thank the other members of her committee: Drs. Bill Carr,

Eugene Sander, and John Zoltewicz for their time and

cooperation.

A very special thanks goes to Dr. Bill Carr for his

honesty and interest and to Dr. Paul Cardeilhac for his

strong shoulder, good ear, and sound advice. Finally, the

author would like to thank Ms. Mary Smith, Ms. Peggy Osteen,

and Mr. Dick Delanoy for their patience and help, without

which it would have been impossible to persevere.














TABLE OF CONTENTS


ACKNOWLEDGEMENTS ii

ABSTRACT v

HOMARINE METABOLISM 1

INTRODUCTION 2

MATERIALS AND METHODS 6

The Maintenance and Injection of
Penaeus duorarum 6

Chromatographic Procedures Utilized
in Homarine Extraction 9

Anion exchange chromatography 9
Cation exchange chromatography 9
Sephadex gel chromatography 11
Thin-layer chromatography 18

Precipitation of Homarine with
Phosphotungstic Acid 20

Isolation of Homarine from
Penaeus duorarum Extracts 20

Procedures Used in the Treatment of
Radioactive Homarine Fractions 24

RESULTS 25

Crayfish Feeding Experiment 25

The State of Homarine in Shrimp:
Free or Bound 27

The Injection of C14-Labeled Compounds 30

DISCUSSION 42








CHEMORECEPTION IN Nassarius obsoletus 44

INTRODUCTION 45

SIZING THE MAJOR RESPONSE-INDUCER(S)
FROM SHRIMP EXTRACT 50

Preparation of Shrimp Extract 50

Ammonium Sulfate Precipitation 51

Ultrafiltration 51

Sephadex Chromatography 53

ISOLATION AND CHARACTERIZATION OF THE MAJOR
RESPONSE-INDUCER(S) FROM SHRIMP EXTRACT 60

Preparation of Shrimp Extract
(Preparation I) 60

Enzymatic Digestion Experiments 61

Two-dimensional Chromatography and
Electrophoresis 64

Electrophoresis and Elution of
Preparation I 68

DISCUSSION 71

BIBLIOGRAPHY 72

BIOGRAPHICAL SKETCH 75












Abstract of Dissertation Presented to the
Graduate Council of the University of Florida in
Partial Fulfillment of the Requirements for
the Degree of Doctor of Philosophy



EXPERIMENTS IN MARINE BIOCHEMISTRY:
I. HOMARINE METABOLISM
II. CHEMORECEPTION IN Nassarius obsoletus

By

Elizabeth Ruth Hall

June, 1974

Chairman: Samuel Gurin
Major Department: Biochemistry

Homarine is endogenously synthesized by Penaeus

duorarum in the free unbound form. The synthesis of ho-

marine in P. duorarum was investigated by injecting shrimp

with a series of C14-labeled compounds. Following injection

of d,l tryptophan (benzene ring-C14(U)), no C14-homarine was

found, thereby showing that tryptophan is not a major pre-

cursor of homarine. Injection of acetic acid-2-C14 did

result in the production of C14-homarine. Previous investi-

gators have shown that tryptophan is not labeled after the

administration of C14-acetate. The possibility that quino-

linic acid undergoes decarboxylation and subsequent methyla-

tion to form homarine was then investigated by the injection

of quinolinic-6-C14 acid. The homarine isolated had a








relatively high specific activity, suggesting that this com-

pound is probably a major precursor of homarine. It seems

likely, therefore, that 1) quinolinic acid is derived from

more than one source in this species and 2) that it may be

produced from an intermediate which can be synthesized from

acetate. A condensation reaction between glyceraldehyde-3-

phosphate and aspartic acid to form quinolinic acid has been

described in higher plants and microorganisms. The incor-

poration of carbon 6 of quinolinic acid into homarine and

the failure of incorporation of C14-tryptophan suggested a

study of aspartate. Although labeled aspartate is defi-

nitely converted to homarine, the radioactive yield was low.

Whether this result was due to major dilution by endogenous

free and bound aspartate is unknown. Finally, the injection

of 1-methionine (methyl-C14) into P. duorarum resulted in
C14-homarine, providing evidence that S-adenosyl methionine

probably contributes the N-methyl group of homarine.

Aqueous extracts of shrimp muscle were fractionated to

determine the size and nature of the major stimulant(s) of

the proboscis search reaction in Nassarius obsoletus. The

size of the major stimulatory molecules) was estimated by

ammonium sulfate precipitation, ultrafiltration through an

Amicon UM 2 membrane, and Sephadex gel chromatography. The

results obtained indicate that the active molecules) has a

low molecular weight of approximately 1000. The activity of

the major stimulant(s) was decreased 70% by aminopeptidase

digestion, suggesting the involvement of a peptide. The








active molecules) was also shown to be soluble in 75%

methanol and insoluble in acetone and chloroform. The 75%

methanol-soluble material was subjected to electrophoresis

at pH 4.8 and the anionic, cationic, and neutral fractions

bioassayed. Of the 3 fractions, the anionic one was the

only one with response-inducing activity. Upon spraying

with ninhydrin, the anionic region revealed 4 ninhydrin-

positive spots. Two of the spots were identified as aspartic

and glutamic acids and shown to contain no activity. The

other 2 spots did contain activity. Upon electrophoresis of

the shrimp extract subsequent to aminopeptidase digestion,

one of the unknown spots was eliminated and the other

diminished in intensity. These results suggest that the

active substance is a low molecular weight peptide that is

anionic in character.






























PART I





HOMARINE METABOLISM













CHAPTER I
INTRODUCTION



Homarine (l-methyl-2-pyridine carboxylic acid) was first

reported in Crustacea by Hoppe-Seyler in 1933 (1). The in-

tervening forty years have brought an elucidation of the

pattern of homarine distribution; but they have yielded little

enlightenment concerning its function, biosynthesis, or

catabolism.

The distribution of homarine has been studied in a

series of animals: basically, it has been found in most

marine invertebrates below the Echinoderms and absent in

terrestrial or freshwater species (2-5). For instance,

Beers (2) estimated the concentration of homarine in the

shrimp, Palaemonetes vulgaris, to be 0.60 1.19 mg/gm of

wet weight; yet, no trace of homarine was found in fresh-

water crayfish by either Gasteiger et al. (3) or Leonard

and MacDonald (5).

Gasteiger et al. (3) have also investigated the dis-

tribution of homarine by tissue in Loligo (squid), Homarus

(lobster), and Limulus (king crab). In general, they found

it had a wide distribution within the tissues of a given spe-

cies with nerve and muscle tissues showing the highest con-

centrations (i.e., 10.3 mg/gm wet weight for the ventral
2







nerve cord of Limulus and 7.6 mg/gm for the cerebral gan-

glia of Loligo). Glandular tissue such as the hepato-

pancreas or gonads contained concentrations nearly as great,

while the skin, mesentery, and stomach contained less. The

blood and urine contained the least (i.e., 0.07 mg/gm wet

weight for the blood of Limulus and 0.038 mg/gm for the

blood of Loligo).

The area of homarine investigation generating the

greatest interest and speculation has been that of homarine

function. The presence of homarine in marine animals and

its absence in corresponding freshwater animals has led to

several investigations of its role in cellular osmoregula-

tion. Levy (4) estimated homarine quantities in nerve cords

of Limulus acclimatized to different salinities, but found

no significant variations in homarine concentration when the

external salinity was varied between 14.3 and 33.5 o/oo.

Similarly, Dall (6) followed homarine concentrations in

blood and whole animal samples from the crab Uca and shrimp

Metapenaeus acclimatized to a range of salinities from 10

to 40 o/oo and found no evidence for a salinity effect. To

date no direct role of homarine in osmoregulation has been

demonstrated.

As a quaternary ammonium base concentrated in nerve

and muscle tissue, homarine has also been suggested as play-

ing a role in nerve function. Gasteiger et al. (3) inves-

tigated this possibility by perfusing lobster heart with

homarine and its likely precursors and breakdown products.







They found that the threshold concentration of homarine

required to alter.the frequency and amplitude of the heart-

beat was 107 times that of acetylcholine required; therefore,

it was concluded to be improbable that homarine had a neuro-

humoral role. Likewise, Keyl et al. (7) found that homarine

did not cause the contraction of the rectus abdominus muscle

of the frog. Welsh and Prock (8) found homarine to have no

observable paralyzing action on Uca pagilator and Kravitz

et al. (9) found homarine to have a negligible neural

inhibitory activity as compared to gamma aminobutyric acid

(GABA) in the crustacean peripheral nervous system.

One other function for homarine has been suggested in

the literature by Haake and Mantecon (10), who propose that

homarine serves as a storage system for C02. They speculate

that homarine is formed by the carboxylation of N-methyl-

pyridinium ion forming a dipolar but neutral ion that

could then be passed outside the cell, decarboxylated,

and returned (by active transport) back into the cells as

N-methylpyridinium. This theory has never been investi-

gated, but appears as an unlikely solution for it would

represent an energetically expensive means of CO2 release.

In 1971, Dall (6) addressed the question of homarine

origin in shrimp. He proposed that homarine is made by the

methylation of picolinic acid derived by the breakdown of

tryptophan. Dall injected each of three Metapenaeus with

10 pCi of C14-tryptophan, homogenizing them (two after 24 hr

and one after 72 hr) in methanol. The methanol extract was








dried, extracted with water, and chromatographed on thin

layer plates. Since radioactivity was found in the UV

absorbing spot corresponding to synthetic homarine,
C14-homarine was assumed to have been synthesized from

C14-tryptophan. However, it is doubtful that this procedure

purified homarine from all traces of tryptophan, thus

casting serious doubt on the results and their interpreta-

tion.

The present work takes another look at the question of

homarine origin. A practical purification scheme for

microquantities of homarine is described and data indicating

that indeed homarine is synthesized endogenously are presented.














CHAPTER II
MATERIALS AND METHODS



The Maintenance and Injection of Penaeus duorarum



The selection of the shrimp, Penaeus duorarum, for these

experiments was based on several factors: 1) they are

readily accessible, 2) they can be maintained in the labora-

tory for an indefinite period, 3) they contain workable

quantities of homarine, and 4) they are easy to inject.

Penaeus duorarum utilized in this study were generally

collected from the Cedar Key area and acclimatized within

the laboratory for at least 24 hr. They were housed in

glass aquaria at room temperature with constant aeration and

filtration of the sea water. They could be maintained for

several months under these conditions on a diet of Biorell

fish food (Sternco).

High survival rates were observed when shrimp were in-

jected either intravenously or intramuscularly. Intra-

venous injections were made through the articular membrane

of the fifth abdominal segment just to the left of the

mid-dorsal line, while intramuscular injections were made

into the ventral portion of the first abdominal segment.

See Figures 1 and 2.




























































Figure 1. Administration site of intravenous injections in
Penaeus duorarum.



























































Figure 2. Administration site of intramuscular injections
in Penaeus duorarum.








Chromatographic Procedures Utilized
in Homarine Fractionation



Of the various chromatographic procedures available to

the biochemist, three were selected for use in this study.

They were exchange chromatogr-phy anionn and cation),

thin-layer chromatography, and Sephadex gel chromatography.

Anion exchange chromatography. At a p1l of 10.9 most of

the dipolar ions present in shrimp extract, including the

amino acids, are negatively charged and thus retained by an

anion exchange resin. Homarine, however, was observed to

pass unretarded through a strong anion exchange column at

pH 10.9. This fact was employed in the purification of

homarine from an alcoholic shrimp extract by chromatographing

the shrimp extract on a 2.5 x 21 cm column of AG1-x8 resin

(OH~ form, 200-400 mesh) that was equilibrated and eluted

with 0.5% NH4OH (pH 10.9). Homarine hydrogen sulfate and

other related compounds were chromatographed as described in

order to ascertain which ones were and were not retained by

the AGl-x8 anion exchange column. The results given in

Table 1 show that the amino acids, glycine and tryptophan,

and the pyridine carboxylic acids, picolinic, nicotinic, and

quinolinic were all retained by the column; whereas the

n-methyl pyridine carboxylic acids, homarine and trigonel-

line passed unretarded through the column.

Cation exchange chromatography. Initial experiments

with a strong cation exchange resin showed homarine to be

firmly bound to the resin and eluted only after the passage
























TABLE 1

THE RETENTION OF STRUCTURALLY RELATED COMPOUNDS
ON AN AGl-x8 ANION EXCHANGE COLUMN


Compound Retained Not Retained


Homarine +

Trigonelline +

Quinolinic acid +

Picolinic acid +

Nicotinic acid +

Glycine +

Tryptophan +








of approximately 15 bed volumes of 0.1N HC1. A 1.5 x 28 cm

column of AG50W-x8 resin (H+ form, 200-400 mesh) was poured

and washed with a minimum of five bed volumes of 2N HC1.

The column was then washed thoroughly with water until the

effluent pH was raised to 6. The sample to be chromato-

graphed was applied to the column and the column washed with

200-300 ml of water and eluted with 0.1N HC1. The eluate

was collected in 6 min fractions of approximately 11 ml

each. The UV 274 absorbance (theAmax for homarine) of each

fraction was measured and plotted against the eluate volume.

A trial run was made with a mixture of homarine

hydrogen sulfate and trigonelline (l-methyl-3-pyridine

carboxylic acid). Trigonelline came off the column during

the water wash whereas homarine was eluted only after the

passage of 800-1000 ml of 0.1N HC1 as shown in Figure 3.

Sephadex gel chromatography. Dall (6) has reported

that the homarine and tissue proteins found in Metapenaeus

blood are inseparable by Sephadex G-10 chromatography. In

an effort to clarify this observation, Blue Dextran

(mw 2 x 106) was mixed first with a sample of homarine

hydrogen sulfate and then with a sample of homarine isolated

from shrimp. Each mixture was chromatographed on a

Sephadex G-10 column (1 x 53 cm) equilibrated and eluted

with phosphate buffer (0.01 M, pH 7.5). The eluate was

collected in 4 ml fractions and the UV 274 absorbance

measured and plotted against the eluate volume. The results

shown in Figures 4 and 5 show that the homarine isolated































3 r-A e-W4-
0 ) M :So0
io rc
0 cj > U W
Orln *r c





o toC o F
to a 4o

Q)

( *-4 0 C)
Ej 4 ) *z 4w-)

S 4J U) rz 4)
.0 r- 0 0
a) .- 0 v-1


0 4J 4-0 A
4 -1 C)



C 4j 4j 0 r0 I
rC)Cv(d 0



C; 0 4-( 0-4 0 0
P 1- .Ca) 4- Q-
4J 0 C)4J4JQ
)-l 0w c U
04 U C -I 0-



C) 0 -I C)4 0 0
bO H 0 (U C)
E-i 3 ttOTi
o O,0 i



) 4O o 0
v4 C) j 0 4 I





































z



aLt-
Li

-I




o













- - - -


r 0 "3DNVt9OSY A

























0)


I:~, c

CO o O






-H o
- ) r-i O

E U OH
CQO I 4W
ro rO .0
2 r o () ;



41 0 0
ON 0o
X2 C : )-




10 C C
o 0 m N 0p
- 1-- I )O





cflo C) -4

r-O -4 a -




COQ Cflr
4-1 S1 ct


;CH 0 04

-m UC U C) 4,
a 11 a)l
*r-i Q m ^
4-1 ~lp ?




































7DNV9dOSqV AAn



























ri n


0. r 0 e

4 0 -
* $-l v.-I
ri C4-i Pc



0C 4 7-

10 0 a 1
4-1 r, P cc
I rC) 4-4

4 1) J ( 0--
0 (U 0

CO! 04*
4- Q)4C
0 CJ j -I

rt ro E R

r- 0


0 0 n o c
0 I 4-1
4-1flop IL











0 a aK al
o CO-i r 0
0 P 4r

l -l 0


C <4- n4-i
4 :0 ( ) (0


o -1 4P,



0 4-u 0 0
qn 04-i




-1 X -4 L zq
Ci C4 it 0 E

















































rW, f7 ZD33NV91OSgV An


.._J
N
D

LL
Lii
-I








from shrimp separated from the Blue Dextran in a manner

analogous to that of the synthetic homarine hydrogen

sulfate.

Thin layer chromatography. Homarine hydrogen sulfate

and a series of related compounds were run on microcrystal-

line cellulose plates (250p) in a variety of solvent sys-

tems. Two solvent systems, 60:20:20 butanol:acetic acid:

water and 90:5:5 methanol:acetic acid:water, were selected

and used throughout this study. The Rf values of homarine

and related compounds in these two solvent systems are

listed in Table 2. In the acidic butanol system homarine

had an Rf value of 0.41 and was well separated from nico-

tinic and picolinic acids but not from trigonelline. In the

acidic methanol system homarine had an Rf value of 0.66 and

was well separated from nicotinic and picolinic acids and

trigonelline.

Chromatographic examination of isolated homarine frac-

tions, using two different solvent systems, revealed only

one UV absorbing spot in each case. The UV absorbing spot,

which had the same Rf values as the synthetic homarine

hydrogen sulfate, gave a yellow color when sprayed with

alkaline a-naphthol (equal volumes of 5N NaOH and 1%

m-naphthol in ethanol) as described by Leonard and MacDonald

(5). No ninhydrin-reacting compounds were detected on these

chromatograms.




















TABLE 2

Rf VALUES OF HOIjARINE AND RELATED COMPOUNDS
RUN ON MICROCRYSTALLINE CELLULOSE PLATES


Solvent System Compound Rf


Ia Homarine 0.41

Trigonelline 0.42

Picolinic acid 0.57

Nicotinic acid 0.73



II Homarine 0.66

Trigonelline 0.54

Picolinic acid 0.78

Nicotinic acid 0.83


aSolvent System I: 60:20:20

bSolvent System II: 95:5:5


butanol:acetic acid:water

methanol:acetic acid:water








Precipitation of Homarine with PhosDhotungstic Acid



Homarine phosphotungstate, a relatively insoluble salt,

was precipitated cold from an acidic homarine solution

(approximately 1N H2SO4) with the addition of 10% phospho-

tungstic acid. This salt, following a wash with an acid

phosphotungstate solution and solvation in 0.5N NaOH, was

reprecipitated by lowering the pH to 1 in the presence of

phosphotungstic acid. The resulting precipitate was washed

and dissolved as before.

Removal of the phosphotungstate was accomplished by the

addition of 10% BaOH to precipitate barium phosphotungstate.

Excess barium, provided to ensure complete phosphotungstate

removal, was in turn removed as barium sulfate by the addi-

tion of 2N H2SO to a pH of 1 leaving a solution of homarine

hydrogen sulfate.



Isolation of Homarine from Penaeus duorarum Extracts



The primary prerequisite of this project was to perfect

a practical purification scheme for the isolation of milli-

gram quantities of homarine from extracts of shrimp muscle.

Fresh shrimp muscle (5-20 gm) was blended 3 times in

100 ml of cold 95% ethanol and centrifuged at 10,000 rpm for

10 min. The residue left after evaporating the combined

supernatants was dissolved in 15 ml of water, shaken with

2 ml of chloroform, and centrifuged. The aqueous phase was








again shaken with 2 ml of chloroform and centrifuged. The

chloroform phases were combined and washed with 5 ml of

water. The two aqueous phases were then combined and evap-

orated to dryness in a rotary evaporator.

The resulting residue was dissolved in 5 ml of water

and passed through an AGl-x8 column at pH 10.9. The

0.5% NH40H (pH 10.9) eluate was neutralized with hydro-

chloric acid, concentrated to approximately 5 ml, and

chromatographed on an AG50W-x8 column. The homarine-con-

taining fraction was eluted with the passage of 800-1000 ml

of 0.1N IIC1 and detected by its UV absorbance as seen

in Figure 3. The eluted homarine fraction was reduced in

volume to 1-3 ml and the homarine precipitated with

phosphotungstic acid as previously described. Figure 6

gives a flow chart of the homarine isolation procedure as

applied throughout this study.

The UV spectra of the isolated homarine hydrogen sul-

fate appeared identical to that of synthetic homarine

hydrogen sulfate where the Amax was 274 nm and the Amin was

243 nm at pH 1. The extinction coefficient (5,11) found for

synthetic homarine (6,200) was thus used to calculate the

concentration of homarine present in isolated homarine

fractions. The isolated and synthetic compounds also gave

the same Rf values when run on thin layer plates in two

solvent systems.



































Figure 6. Flow sheet illustrating the fractionation pro-
cedures utilized in the isolation of homarine from shrimp
extracts.












Shrimp Muscle

blend in cold 95% CH30H


Insoluble Soluble
dry, extract with H20


Insoluble Soluble

shake with CHC13


CHC13 phase H20 phase

apply to AG1-x8 column
wash with 0.5% NH40H


Retained Unretarded

apply to AG50W-x8
wash with H20


Retained Unretarded

Salute with 0.lN HC1


Retained Eluted
phosphotungstate
ppt.


Precipitate Supernatant

Dissolve 0.5N NaOH
add 10% BaOH, 2N H2SO


Precipitate Homarine








Procedures Used in the Treatment of
Radioactive Homarine Fractions



Isolated homarine fractions ranging from 0.1-0.5 ml in

volume were counted in a Beckman LS 230 liquid scintillation

counter using a toluene based cocktail with 10% v/v of BBS-3

and 0.3% wt/v of TLA fluor (12). Background counts for

0.1-0.5 ml of synthetic homarine hydrogen sulfate (1 mg/ml)

were determined to be 31 2 cpm for the C14 ISO-SET, with a

calculated counting efficiency of 91%. All samples were

counted for a minimum of 5 10-min counts and the average cpm

calculated.

Aliquots of active homarine fractions (fractions having

greater than 10 cpm above background) were streaked on 500p

microcrystalline cellulose plates and run in two solvent

systems. The homarine band was scraped from the plates and

extracted with water. Estimated specific activities of the

chromatographed homarine samples were calculated and com-

pared to that of the original homarine fraction.














CHAPTER III
RESULTS



Crayfish Feeding Experiment



The question of whether homarine is synthesized by

shrimp or merely ingested and stored was originally

approached by feeding shrimp a non-homarine diet while

monitoring their homarine content.

Twelve shrimp were maintained on a diet devoid of

homarine by feeding them frozen crayfish (Cambarus sp.) for

21 days. At 7-day intervals, 3 shrimp were killed and their

homarine content estimated. The values obtained for the 3

samples from each group were averaged and the averages com-

pared. As seen in Table 3, greater variation was observed

in the homarine concentrations of the shrimp within any one

group of samples than between the average concentrations of

the different groups.

Since the homarine content did not significantly de-

crease over this 3-week period, it was considered quite

probable that homarine was being synthesized by the shrimp

rather than being obtained from the diet.
























TABLE 3

HOMARINE CONCENTRATIONS FOUND
IN CRAYFISH-FED SHRIMP


Homarine Concentration (mg/gm)
No. of Days Shrimp Shrimp Shrimp Average
on Crayfish Diet 1 2 3


0 0.98 0.59 0.50 0.69

7 1.11 0.37 0.55 0.68

14 1.16 0.79 0.53 0.83

21 0.51 0.76 0.30 0.51








The State of Homarine in Shrimp:
Free or Bound



Encouraged by the results of the crayfish feeding ex-

periment, a series of injection studies with C14-labeled

precursors were planned. However, before any labeling

experiments could be done, it was necessary to determine the

state of homarine (free or bound) in the shrimp.

Dall (6) has suggested that homarine appears bound to a

small peptide in the shrimp, Metapenaeus. This question was

investigated in Penaeus duorarum by considering each of 3

possibilities: 1) that homarine is bound to an alcohol

insoluble peptide or protein, 2) that homarine is bound to a

small alcohol soluble peptide, and 3) that homarine appears

in the free state.

Fresh shrimp muscle was blended 3 times in cold 95%

ethanol and centrifuged at 10,000 rpm for 10 min. The

ethanol-insoluble precipitate was hydrolyzed in 6N HC1 at

1000C for 28 hrs. The hydrolyzate was then chromatographed

on an AG50W-x8 cation exchange column. The eluate was

collected in 6 min fractions and the UV 274 absorbance

measured and plotted against the eluate volume. The UV 274

absorbance shown in Figure 7 within the region of homarine

elution (300-1000 ml) represents less than 1% of the

homarine subsequently recovered from the alcohol-soluble

fraction.

The ethanol-soluble fraction of the shrimp extract was

then evaporated to dryness. The residue was thoroughly




















b0


r-I C0) r
*Ho a Cd
,-i o .,I



r o 4-
0 a) O





H cH -4
C ( 0 00







,-. w 0 -, c
-1 r rl



N CU 4 0- 0
tO ,-t 4 -
-10 0


4 ., ,C- 0.

hou Dc
0-I 0 C
O-4 Cd H -t






o c 0 0 )C
-0 cri 0 C
*0 *r-I -4 X
0o a)40 r

oC) o 0-i



Nr 0- 00


tO n- > 0 r0
C: E 41 l a)C





'-0 ; H C C)
O C 0 4JO
oC C 0

0 W) 4 C
'-f U C( 0 0
HOC) CJ






d Or-I -- bZ '4-
$ C4 C

r C Irtl C) '-


d CDC) C) 004






29















o






0
3-





o








._J
0




/ --


oi

LU
-I






0





o








extracted with 10 ml of water, of which 1 ml aliquots were

chromatographed on an AG50W-x8 column before and after acid

hydrolysis. Theoretically, free and bound homarine should

have different chromatographic properties, such that if a

homarine-peptide were hydrolyzed, the eluted homarine peak

would be increased in proportion to the quantity of bound

homarine present. Yet a comparison of Figures 8 and 9 shows

that the homarine peak is not increased upon hydrolysis.

The amount of homarine eluted from the column was 0.38 mg

from the unhydrolyzed fraction and 0.34 mg from the

hydrolyzed fraction.

Thin layer chromatograms of homarine isolated from

shrimp were also run before and after acid hydrolysis. Both

chromatograms had a single UV absorbing spot and neither

contained any ninhydrin-reactive material. Furthermore,

synthetic and isolated homarine fractions exhibited identi-

cal chromatographic properties on a Sephadex G-10 column as

seen in Figures 4 and 5.

The evidence presented here strongly suggests that

most, if not all, of the homarine exists in Penaeus duorarum

in its free molecular form.



The Injection of C14-Labeled Compounds



Additional evidence for the synthesis of homarine by

shrimp was obtained by injecting C14-labeled compounds into

the shrimp either intravenously or intramuscularly with the




























rH

r-1 C




o U
Cd rdM
OUO
4 0 E




V 0
1 *1 0


ci bO

00

0 (1

CO
-4 0E
co c

ed -4 *





,(l CIO
0 o*





0

O
OC








U V
00-1 H

oa) 4


.-4 .


I rd
*rJ (

E- cS rd
*i- *U








































rVW frL ai33NvgJIOSgV An



























C
0
.-I
"o

rN




C; 0





(4 1-1
Hr --
00 a)l






0c 0
CD

M O




0 0
OO




co
- -4d
4 0
PO CO





S*rd m
, (0) Cd








C)
P 4








co
r-4-
0 0-'












6 *- I
( *l 0 C



-H 4jM
































Z

03

SLL
LL


4-












St

I3NVBOSgV An








subsequent isolation of homarine, Whenever C -homarine was

isolated, it was considered to have been synthesized from

the injected C14-labeled compound. When C14-homarine could

not be isolated after the injection of a C14-labeled com-

pound, that compound was considered to be of little impor-

tance in the derivation of homarine.

C 4-tryptophan. The close chemical relationship of

homarine to nicotinic acid and its N-methyl derivative,

trigonelline, has led to suggestions that homarine is a

product of tryptophan catabolism (6). Five pCi of

d,l tryptophan (benzene ring-Cl4(U)) were injected into the

vascular system of shrimp and a crude homarine fraction

isolated 23 hrs later by thin layer chromatography. When

this sample of homarine was rechromatographed with cold,

carrier tryptophan the calculated specific activity was

only 1/4 that of its previous value. Thus it was apparent

that the homarine fraction being counted was not pure

homarine; therefore the experiment was repeated after the

development of an improved purification scheme for homarine.

This time ten pCi of d,l tryptophan (benzene ring-C14(U))

were injected into three shrimp. Two shrimp were injected

with 2pCi each and killed after 6 hrs. The third shrimp was

given two 3pCi-iniections 12 hrs apart and was killed 12 hrs

after the second injection. Alcoholic extracts of the 3

shrimp were combined and the homarine isolated. Although

7 mg of homarine were obtained from the C -tryptophan in-

jected shrimp, the isolated homarine fraction contained no








counts above background. See Table 4.

Isolated and synthetic homarine fractions gave the same

Rf values in two solvent systems and had identical UV

spectra.

C14-acetate. Having ruled out tryptophan as a major

precursor utilized by the shrimp in homarine synthesis,

C14-labeled acetate was employed in an effort to obtain

C14-homarine and thus provide additional support for the

thesis of homarine synthesis by the shrimp.

Two 62.5pCi-injections of acetic-2-C14 acid were given

12 hrs apart. After twelve additional hrs the shrimp was

killed and its homarine isolated. The 4.5 mg of homarine

isolated were found to contain a total of 2220 dpm.

Aliquots of this homarine fraction run in the acidic butanol

and acidic methanol solvent systems retained their activity

as seen in Table 4. The estimated specific activities of

the homarine run in the acidic butanol and the acidic

methanol solvent systems were 85.4% and 98.6% of the

specific activity of the original fraction. The fact that

C14-homarine was isolated from shrimp injected with

C14-acetate provided additional support for two points:

1) that shrimp do in fact synthesize homarine, and 2) that

tryptophan is not a direct precursor of homarine, since

tryptophan is not labeled with the injection of C14-acetate

(13).
C14-quinolinic acid. The possibility that quinolinic

acid undergoes decarboxylation and subsequent methylation in













0 0

Oci 01-I
a, a3






4J )*4 u
.0-< 0





r4

U L














-4
0








0ci0
Ix





O>








po




01
E













Oi
to>



0
o




















C




4
0

0
to
0)rp

J3





nl

1-


o

0
4

O
m l



O H


HM





0
E-0
H p

4









oL
OH




SH
HZ


41
SQ




H

HE





Hm


I cq r- r- -w
I 0N CI








co
r-I 0 N 0; cl
4 0 10 in
r-4










CM




o l 0 0 0
H CM r-I CM 0
v-l -I
-4 -L4





I


-4 0 "
4 rH -I I

I0 5 *4 Id
et co 0 -H
ce I 0



0> H I 0z
0 *H O -4




QC Cl !- 4-I 0
>5 0r .0
C U -1 C U) c0





C 4 r-I r -

-h O 0 I I i
:Jr


\0 co
0 Cl
Ho rn
-- m






CM 0
C4 4
-4


bD
0

o
0
4-

0
4 w
SQ)

C 0
o -









O 0
C o W





a O*






cc ci
0 0 1
44- 4





0 *-l n





4 0 o
-4>












.w 4J r-I
O O C


















o -nl T-I
4- 0









0 0

SU4-
(U n















r o
co
n CD

*H










0 O

0 4-
>4 .0W
i-l ( 0 n



4 ( 0




















:J
o >4,

-5 > 0

a1 4u-






a
*r4 0 0
U) UO
ci U)
to



*H -




4-ici





t0 r 0


0
a


Lc
.0
o
0


0
0









U)
dce
C)




00



O-HU)







O O
ou
4Q) .
,i -
CIr-I







00


* r II



um
0

















ao
-) -0-









cHm
-e,

00
.-0



QC
-4i
4W1











U)
00
14-1 -



















0 0

Ec
F01
41 0
oIn





0 t


















1-1 e
00o








shrimp to form homarine was investigated by injecting 15gCi

of quinolinic-6-C14 acid into Penaeus duorarum. Twelve hrs

later the shrimp was homogenized. The isolated homarine

fraction had an estimated specific activity of 1127 dpm/min.

Subsequent chromatography in the acidic butanol and

acidic methanol solvent systems did not decrease the activ-

ity of the homarine fraction as seen by the estimated

specific activities of these fractions listed in Table 4.

The fact that carbon 6 of quinolinic acid is incorporated

into homarine supports the proposed pathway shown in

Figure 10. Furthermore, the high specific activity of the

isolated homarine fraction suggests that quinolinic acid is

an important precursor of homarine.

C14-aspartate. Leete (14) and others (15, 16) have

proposed that quinolinic acid is formed by a condensation

reaction between glyceraldehyde-3-phosphate and aspartic

acid in higher plants and certain microorganisms. See

Figure 11. The incorporation of carbon 6 of quinolinic acid

into homarine and the lack of incorporation with C14-trypto-

phan made this pathway an attractive possibility.

One shrimp was injected with 25pCi of 1-aspartic

acid-C14(U) and killed 9 hrs later. The homarine isolated

from the shrimp had only 121 dpm in the 16.9 mg isolated, or

an estimated specific activity of 7 dpm/mg. The homarine

fraction retained its activity after chromatographing it in

two different solvent systems as seen in Table 4. Apparently

C4-aspartate can contribute carbon atoms to homarine, yet

not as readily as C14-acetate.












0 7

IIO
2 -OH

10


Quinolinic Acid


I 0
Ni" -OH

Picolinic Acid


SCH,


-0


H+
Homarine


Figure 10. A proposed pathway for the incorporation of
carbon 6 of quinolinic acid into the homarine molecule.













O
H 2C

4CHOH
51
P-O-CH2
Glyceraldehyde
3-Phosphate

+

2CHT,-COC

'CH-'COO


NH,
Aspartic Acid


OH
7
OH. COOH
4 2


N OOH
)H N
1,2 dicarboxy-3,4 hydroxy-piperidine
H



3 7
COOH
4 2


Quinolinic Acid


Figure 11. Biogenetic scheme for the formation of quinolinic
acid in higher plants and some microorganisms as proposed by
Leete (14) and others (15, 16).








C 4-methionine. In order to determine whether the

methyl group of homarine is derived from methionine, two

shrimp were given intramuscular injections of 50pCi of

1-methionine (methyl-C14). The shrimp were homogenized

after 12 hrs and their homarine extracted. The 3.2 mg of

homarine isolated contained a total of 557 dpm. Aliquots of

this homarine fraction run in the acidic butanol and acidic

methanol solvent systems retained their activity as seen in

Table 4.

The fact that the C14 from the methyl group of

methionine was incorporated into homarine provides strong

support for the suggestion that S-adenosyl-methionine

contributes the N-methyl group of homarine.













CHAPTER IV
DISCUSSION



Evidence has been presented which demonstrates that

homarine is endogenously synthesized by Penaeus duorarum and

that most,-if not all, of it exists unbound as free homarine.

Tryptophan is known to give rise to nicotinic acid,

which is closely related to picolinic acid, via a quino-

linic acid pathway. Thus, it has been tempting to assume

that homarine is produced by essentially the same pathway.

However, the results given indicate that tryptophan is not

an important precursor of homarine, for not only did injec-

tions of C14-tryptophan yield inactive homarine, but labeled

acetate was converted to labeled homarine; and Cowey and

Forster (13) have shown that tryptophan is not labeled

after the administration of C14-acetate.

Results obtained by the administration of radioactive

quinolinic acid suggest that this compound is probably a

major precursor of homarine. It seems likely, therefore,

that l)quinolinic acid is derived from more than one source

in this species and 2) that it may be produced from an

intermediate which can be synthesized from acetate.

There are very few metabolic pathways known to give

rise to quinolinic acid. Mention has been made (14-16) of

42








a condensation between glyceraldehyde-3-phosphate and

aspartate which gives rise to quinolinic acid. Although

labeled aspartate is definitely converted to homarine, the

radioactive yield was low. Whether this result is due to

major dilution by endogenous free and bound aspartate is not

clear.

Since labeled acetate appears to be readily incorpor-

ated into homarine, it will be of interest to test other

metabolites that may be derived from acetate: pyruvate,

short chain tatty acids, members of the tricarboxylic acid

cycle and the non-essential amino acids. Although it would

appear to be highly unlikely, there is always the possi-

bility that acetate may condense with a nitrogen-contain-

ing metabolite derived from one of the essential amino

acids.

Finally, these experiments indicate that homarine is

derived by decarboxylation of quinolinic acid followed by

subsequent methylation of the ring nitrogen. It is

probable that the latter reaction occurs via S-adenosyl

methionine.





































PART II






CHEMORECEPTION IN Nassarius obsoletus


__













CHAPTER V
INTRODUCTION



Chemical attractants which may act over long distances

to orient an animal toward the apparent source of those

chemicals are of widespread importance in food localization

(17). For example, turkey vultures are attracted and will

orient to ethyl mercaptan dispersed in the air by a fan and

it has long been known that sharks are attracted to very low

concentrations of vertebrate blood. Chemical attractants

that act over long distances must be freely diffusable in

the environment of the animal that is to be attracted (i.e.,

attractants for terrestrial animals must be volatile and

those for aquatic animals water soluble).

Although some basic work has been done on chemorecep-

tion in marine invertebrates in general and gastropods in

particular, any understanding of this phenomenon at the

molecular level awaits the identification of the compounds

involved (17, 18, 19). The majority of chemoreception

studies have been oriented along three major lines of inves-

tigation: 1) proof that observed responses in certain

animals are chemically induced, 2) investigations of the

chemical nature of attractants, and 3) tests of a spectrum

45








of known compounds for their stimulatory activity. Then in

1967, Carr made a significant attempt to account for the

responses of the marine mud snail Nassarius obsoletus to

shrimp extracts, both in terms of the compounds present and

their relative concentrations (20,21).

Nassarius obsoletus is particularly suitable for chemo-

reception studies, as it displays a stereotyped response

(i.e., extending its proboscis) which is convenient for

measuring the effectiveness of stimulatory substances.

Using the proboscis search reaction as described by Carr

(20), Gurin and Carr (22) were able to show that the stimu-

lation induced by human serum and by oyster mantle fluid

was attributable primarily to very low concentrations of

specific proteins. In serum the major stimulant was highly

purified serum albumin (ca. 10-9 M), whereas in oyster

fluid the major stimulant proved to be a homogenous

glycoprotein (ca. 10-10). This glycoprotein accounted for

more than 90% of the stimulatory activity of the oyster

mantle fluid. This was the first time that an attractant

had been isolated from an animal fluid and shown to account

for essentially all of the activity of the natural fluid.

Carr et al. (23) screened biological fluids and

extracts from eight species of marine animals to determine

the nature of the principal inducers of stimulatory

activity in Nassarius obsoletus. The major response

inducers from the scallop, clam, blue crab, sea urchin, and

three fishes proved to be macromolecules that were ammonium


I









sulfate precipitable, non-dialyzable, and retained by ultra-

filtration using an Amicon UM 2 membrane. In contrast,

analyses of various fractions obtained from shrimp extracts

show that their major response inducers are low molecular

weight substances which are dialyzable and are included in

the bead matrices of Sephadex G-10 columns which will

exclude globular molecules with molecular weights of 700 or

more.

A variety of low molecular weight substances, such as

amino acids, betaines, and amines, identified in shrimp

extracts have been tested for their stimulatory activity

with none of the isolated substances singly or in mixtures

eliciting as strong a response as the original extract (21).

Glycine, the most active of the compounds tested, did

possess marked stimulatory activity in solutions of 10-3 M.

Considering the evidence suggesting that response-inducers

are often proteins (22, 23, 24), a series of glycine pep-

tides were assayed for activity. However, as can be seen

in Table 5, glycine proved to be at least ten times more

active than any of the peptides tested.

An exciting possibility emanating from the work of Carr

et al. (23) is the probable presence of a response-inducing

low molecular weight polypeptide in shrimp extract. If

such a polypeptide were isolated and sequenced it would allow

the analysis of chemorecption in Nassarius obsoletus at a

molecular level. The present work represents a joint effort











(12



m
0
Q.











uv














v



Cd
4J
a()

















Scd
s-H


2:

0
























r- <


C' CO m C) 0
-4 r-I CM Cn) m


cl ,0
rA cv
Hu *-P *.-= 0
0 u U *r-

:>. bO i

60 H 4I Cd

-ri aa ( a
- cv Cv c


L 0 0 0 0 0 U O 0 0 0 0
r- uLr Y I4 C' -I -A












0 0 0 0 0 0 0 0 0 0 0 0 0
CN N C r-i C r-I c -i N -A N r-4 -4 r-4












N Cl C'l c') C14 cn C1 cq C' CN Cn) C4
I I I I I I I I I I I I
o oo o o0 o o0 o o 0
-- 1 r- r- 1 r--I r--I -- --4 r-4 r-4 r-4 -4 -4


co


r-4
1-4 0
Cd 4J
--I

HO

4-4 C0
4d Cd







0 0
Cd r
O C

bo
ci a

u d
! a

Z 4



Ca Q



4-J


0
> m
QU
cc
cd
td *r-
-a
a


o r(

rl Cn

3 (


d ,


r- Co) CM
-4 C- C14
r-4 r-4 CM4


1-1






0


r-4
c >c

r-I w
U M



cv r-
U :,
I 40









by William Carr, Samuel Gurin, and Elizabeth Hall to isolate

and characterize the major response-inducing molecules)

from shrimp extract.













CHAPTER VI
SIZING THE MAJOR RESPONSE-INDUCER(S) FROM SHRIMP EXTRACT



There are a variety of techniques available to the bio-

chemist for approximating the molecular weight of specific

substances. Several of these techniques were employed in

estimating the size of the major stimulatory molecules)

found in the muscle extracts of the shrimp, Penaeus

duorarum, specifically, ammonium sulfate precipitation,

ultrafiltration through an Amicon UM 2 membrane, and

Sephadex G-25 and G-10 chromatography.



Preparation of Shrimp Extract



Aqueous extracts of the shrimp, Penaeus duorarum, were

prepared by gently shaking coarsely minced shrimp muscle

with 3 volumes of cold water. After shaking for 30 min in

an ice bath, the solution was centrifuged for 30 min at

10,000 rpm in a Beckman J-21 refrigerated centrifuge. The

clear supernatant was decanted and tested for activity. The

solution was highly stimulatory with only 0.16 jl of solu-

tion per ml of sea water necessary to induce the proboscis

search reaction in 50% of the test animals (effective dose

for 50% of test animals = ED50).

50








Ammonium Sulfate Precipitation


Eighteen ml of saturated ammonium sulfate (0.7 g of

ammonium sulfate per ml of water) were slowly added to 2 ml

of prepared shrimp extract and allowed to sit overnight at

70C. The resulting 90% ammonium sulfate solution was then

centrifuged for 30 min at 10,000 rpm at 0C. The precipi-

tate was washed with saturated ammonium sulfate solution,

redissolved in two ml of water, and tested for its activity.

As illustrated in Table 6, only 18% of the biological

activity was precipitable in this manner suggesting that

either the major stimulatory factors) in shrimp extracts is

non-protein in character or is of low molecular weight.

Similar experiments were run yielding comparable results.



Ultrafiltration



Another portion of the prepared shrimp extract (38 ml)

was ultrafiltered through an Amicon UM 2 membrane at 40C and

35 psi of nitrogen to a retenate volume of 4 ml. The

retentate and ultrafiltrate were brought up to the original

volume of 38 ml and bioassayed. Table 6 shows that the bio-

logical activity was rather evenly distributed between the

retentate, with 57% of the original activity, and the

ultrafiltrate, with 35% of the original activity. This

indicated that the major response inducer(s) is within the

threshold range of the pore sizes of an Amicon UM 2

























o co (o io
,-l















o co N t
rD CO CO O


E-4
X


WHO
SPE-l
U H
-HH

M H
WHZ


0 0-








0
I-4
< M





HH



0 C-




O

--i


4-1


)
> -
04
Uo J

cr


I 4-
Q 0O

O hh
S *H k4
4J 4-J O) -1
0 >0 F>
l4J 0 O0
O 1 0 4J
0 I C)4 <
C) 4-4) Q<

-H o 0 o






-r4 J0 U


V 400-
w JU) QW

0E rA 04-J

r 0 0 0 0

0 H) Ot) 0
U) 0 -0 0
>a 1 -Ho 0Q



>0C 0a40 0
O 0 C4- U 0
0 m aQa-) -










I0 ) ,'- 0 j
n -.- 4J 4 4n-4
[0 4D O 0



0 1C) 0) C)'C




o c0 0a
C0 0 p wH -4

L)- C*) 00 f
C) .Co -r-i r
C (B >, C 41 iv











c3 ^h >- ct 4J)


0


X 4





0
H4J





0, 4J
E-- 4








membrane. Such substances would have a molecular weight of

at least several hundred and probably less than two thou-

sand. Additional ultrafiltration data further indicate

that the molecular size of the major response inducer(s) is

within the threshold range of the membrane.



Sephadex Chromatography



In an effort to further isolate the stimulatory

moleculess, a more concentrated shrimp extract (prepared

from 1.5 volumes of water per volume of shrimp) was chroma-

tographed on a Sephadex G-25 column, which will fractionate

molecules ranging from 1,000 to 5,000 in molecular weight.

Ten ml of shrimp extract were applied at room temperature

to a Sephadex G-25 column (5 x 30 cm) that had been swollen

in phosphate buffer and equilibrated with distilled water.

The shrimp extract was eluted with distilled water at a flow

rate of 30 ml/hr. The eluate was collected in uniform sam-

ples (100 drops each) and the UV 280 absorbancd recorded as

seen in Figure 12. Individual samples were pooled to make

four fractions as indicated in Figure 12. Each fraction was

lyophilized, redissolved in 10 ml of distilled water and

bioassayed. The concentration of protein in each fraction

was estimated by the procedure of Lowry (25). Fraction I

corresponded to the void volume as determined by chromato-

graphing Blue Dextran (mw = 2 x 106). The included frac-

tion, Fraction 3, contained 89% of the activity present in






















0 0 *0
4 4 0
0 U *-l

4J r-1 4 Wi

0 nrdo
M 000) Oo

1 0 () 0 0
c ed ,o b) i


4tH C)
&d OJ I
CH (a Q l 41
Cr 4 : a) mO

-o 0 0o
L) 4- 4-1 0 C 4-
0 0 0 0 4U




O n -44tH 0-
(0 420 () 4 f.
0l ) r r- -d C







-4 tr4N 4 o C-4 0 )
41 P ci -
na eD m -i C -4




0i c 0)> > 4
40 co0 On o
0O 5 4r r nj r-4
kCOMr-I 42




0d 0-1 0 0 t
Orl O C O







r40 4J 0
) X C -> 4 C*






-4 4-) C O3
I Q)i (Ii 4J 0


0 0 'H 0 0) 0
(_) ~ i c to o < 4






I ( j3 -U-W C'l
CN ') a-1420
(U U4rl cU*)J

O '0 U -) *Ii 4 4J

0 -CO Cr- 9
*00U000 -
an e O 4 n3 r-





















0





N0




CO

Lr
bJ



cD

r(1




o


o O J o co
-- C 0 0
TvL 09z -ONV"dOV An








the combined Fractions 1 through 4 (see Table 7). However,

Figure 12 illustrates the fact that the column was over-

loaded, thus there was no fractionation of the included

substances.

In order to obtain additional information on the

molecular size of the stimulant(s), 2 ml of shrimp extract

were similarly chromatographed on a Sephadex G-10 column

(2.4 x 47 cm). Four ml samples were collected and analyzed

for their UV 280 absorbance. The samples contributing to

each of the three UV 280 peaks shown in Figure 13 were

pooled to make three fractions. Bioassaying the fractions

showed 56% of the original activity was recovered in

Fraction 3, the included fraction, with very little activity

found in Fractions 1 and 2. An amino acid analysis of

Fraction 3 showed a significant concentration of amino acids

present. These results indicate that the stimulatory

molecules) is of low molecular weight and cannot be readily

separated from the amino acids present by Sephadex

chromatography.














*0 >

Q) >
> .,-4

o o


4-:
9O 0


0 -4 0 I e -4
o O i-I i r~
r I











So 4 1 or











ND oC in4 i o
CN O Lf) co Li 1-4

, C") 0C C, N -4"












r'N cO O ) ,-rl













0
a) u

,-4 U
.
1--4







E t
CE
co (H 4 N cq '.0
0
*r
S 0 0 0 0



r4- X Cu Cud Cu LC
C ) ) r) U U u

O g [| c f| [


lu
o

0




OW
0



-4





OO
S *r-1
.0

biD O








0 NO
S ,--4
0 0










a ma
40 *-1 '-4





O 0 0
S4- 0







cuu f




'- .-W
4 -0
(ti Cui


































)O -r-
Od *-
*lH
*r 4 0












0 0 l O






>u-- e 0 -
0 m
C) 44Q)W
co 0













,0 c 0
.H U u





0 N4




Q) 4-U





Hl g- a)14
nj .Q u o


0
4-J
O





0







0
ed












On
4J-
0
.H

1-
* 00
















0
4)

Cd
r4I






















o "o d '0 o

0 0 o
U a) 2 04

Ca o r


C)/3 r- CO ) 4- CO

cd 3 ico
0 N4



0 :) 0 "
C bCd
o n o--o
a 00 4
S) )o0 4u

H54 0 ,> 4.
+-P P U 3 MI-4
4 4-1 ,) Z-C. "
0) Cd 0 .0
-0 CoU 0)


0 0) 00 4




O-l D.4 O
442 4- 4(


0 0
0 U4-1 4- 0
o o 0,A
0H 10 0
0 03 2 -0
-0 4 r0 -42
nj( 4J r-



0C 4200t
0 a) 0 0n *H

::3 4-) CO nd





4 r- C Ma




-'-1 022 U) 1
* 0 42 H Cur-4
LL,0 4 il n il





59












o

O







7 z
t



uJ












33N C An
SDNVfO S'V Afl


M -
'n 0 .Z













CHAPTER VII
ISOLATION AND CHARACTERIZATION OF THE MAJOR
RESPONSE-INDUCER(S) FROM SHRIMP EXTRACT



Preparation of Shrimp Extract (Preparation I)



Several preliminary experiments were performed to de-

termine whether the stimulatory activity of aqueous extracts

of shrimp could be precipitated with methanol. Freshly

minced shrimp muscle (50 gm) was stirred 1 hr at 30C in 2%

NaC1 solution (50 ml). After centrifugation cold methanol

was added to the clear supernatant to a final concentration

of 75% methanol (v/v). The resulting precipitate was

chilled in a deepfreeze overnight, collected by centrifuga-

tion, and bioassayed. The precipitate was found to contain

no more than 10-15% of the original activity, while the

75% aqueous methanolic extracts usually contained 80-90% of

the original activity. The residue, left following evapora-

tion of the 75% methanol, was washed with absolute acetone

and then extracted repeatedly with cold absolute methanol.

When the methanol had been evaporated and the residue

dissolved in water it was found to have 70-75% of the

original activity. Several such preparations were combined

to yield 20 ml of a clear, slightly yellow solution. This

solution was vigorously shaken with 3-4 ml of chloroform for

60








a few minutes and centrifuged. The aqueous fraction was

collected and aerated for several minutes to remove residual

chloroform. All color was removed by the chloroform; never-

theless the clear aqueous phase was filtered by gravity

through a small wet filter to remove any denatured insoluble

protein. The resultant solution which retained full activ-

ity (preparation I, ED50 = 0.06 pl/ml) was used for all

subsequent studies. See Figure 14.

An amino acid analysis of preparation I by the Stein-

Moore technique indicated the presence of trace quantities

of most amino acids with glycine, arginine, alanine, serine,

and proline present in significant amounts.



Enzymatic Digestion Experiments


In order to determine whether the shrimp stimulatory

factors) has the properties of a small peptide, a series of

enzymatic digestion experiments were performed. Aliquots of

preparation I (0.6 ml) were diluted to 5 ml and incubated

respectively with trypsin, carboxypeptidase, and

aminopeptidase at pH 7 for 1 hr at room temperature. All

enzymes were highly purified Worthington preparations.

After incubation each solution was shaken vigorously

for 2-3 min with 1 ml of chloroform. The suspensions were

allowed to settle and the supernatant fluid filtered by

gravity through a small wet filter paper. The chloroform

phases were washed several times with 1 ml of water and

refiltered. The aqueous fractions were collected and

































Figure 14. Flow sheet for the fractionation
of preparation I.









Minced Shrimp Muscle

extracted with cold 2% NaC1


Insoluble Aqueous Extract

add cold CH30H to 75%


Insoluble Soluble

evaporate to dryness

Residue
Swash with acetone

Soluble Insoluble


Insoluble


Extract with cold CH30H


Soluble
Soluble


evaporate to dryness


Residue


dissolve to H20


Insoluble Soluble
shake with
CHC13

CHC13 phase H20 phase
filter


Preparation I
Preparation I








aerated until the odor of chloroform could no longer be

detected. Each fraction was then adjusted to 10 ml and

bioassayed. The results seen in Table 8 reveal only a

slight decrease in activity after trypsin digestion; how-

ever, digestion with aminopeptidase resulted in a 70%

decrease in biological activity. This suggests that the

active principal is a peptide.



Two-dimensional Chromatography and Electrophoresis



A sample of preparation I as well as preparation I

which had been previously digested with aminopeptidase were

then concentrated in vacuo and spotted on a large sheet of

Whatman 1 paper. Two dimensional chromatography and

electrophoresis were then employed. Vertical chromatography

was performed using a 4:1:5 butanol:acetic acid:water sol-

vent system; subsequent electrophoresis of the sheet was run

in pyridine acetic acid buffer (pH 4.8) at 2000 V for 1 hr.

Upon staining, preparation I revealed four distinct spots in

the anionic region; two spots were identified as glutamic

and aspartic acids and the other two were unknown sub-

stances. The sample of preparation I previously digested

with aminopeptidase lacked one of the unknown spots, with

the other unknown diminished in intensity. These results

suggest that the active substances) is a peptide which is

anionic in character. See Figure 15.






















So 0 0
















0 CO 0 CI

0 0 0 0


(U











O
0 u


S 0







rz\

-4


'C


























M


.a 0





S,0
+ +


O H






0 0
ni ca


aa
.r
H >




* 0



0
0 (






0





















NO
-r-4 a) 'a
0 03C c 0
Pr-4 *-H m 0

,r cd 0 L
a*l n c *i

0 *H r--C 0 4 0Z

ao t-lr r-4
-4 -r- -, 0 CO CC

o0 U Uo

,C 4 -1 4
OW COCO
C ,O b C

-r -1 O v
. 0 r4 0

" -O *Q r'

0 a 0 P0 W
00 *-0 4
l -r- o 0 l -

0 CQ 04
O'-''4-4 04Z


4 0 0ft



0 0 07-0
2- 0 4 0
& oU (a)









0 *Cr uH 0442
*-) 4 COO


) 04 C 0

0 *0424
00 0 C 0
Z 0) "- *C 0


*1 0 m -0 0 C0
z0 -H C OOr--
- 0 -000

4 rd Q) ) l


*H- 0 0 0C
C1 444 Cd O' 0 CO



























DN


ri 1.
MFIF








Electrophoresis and Elution of Preparation I



To provide additional evidence that the stimulatory

substances) is indeed anionic, the anionic, cationic, and

neutral fractions were eluted and bioassayed. A large sheet

of Whatman 1 paper was spotted with six spots of preparation

I (0.2 ml each) and subjected to electrophoresis at 2000 V

in pyridine acetate buffer at pH 4.7 for 1 hr. The paper

was then cut into six strips, each corresponding to one of

the spots of preparation I. The top and bottom strips were

stained with ninhydrin to show some amino acids and pep-

tides. See Figure 15. Two of the remaining strips were

cut vertically to separate the anionic, cationic, and neu-

tral fractions. Each section was separately eluted with

50 ml of millipore-filtered sea water by passing the eluting

fluid dropwise through the strip. As seen in Table 9, the

anionic sections yielded stimulatory solutions, 50% of the

original activity one time and 33% the other time. No

activity was recovered from the cationic and neutral

fractions.

The final two remaining strips were combined and cut

vertically to separate the four anionic substances seen in

Figure 15 and designated as follows: peptide 1, glutamic

acid, peptide 2, and aspartic acid. The strips containing

peptide 1 and peptide 2 were separately eluted with 50 ml of

millipore-filtered sea water and assayed. Peptide 2 was

quite active, accounting for 65% of the original activity;




















TABLE 9

THE RELATIVE ACTIVITIES OF ELUTED FRACTIONS
AFTER ELECTROPHORESES OF PREPARATION I


% Recovery
Fraction ED50 (Il/ml) of Activity


Preparation I 0.065 100

Cationic ----- 5

Neutral ----- 5

Anionic 1 0.13 50

Anionic 2 0.20 33

Peptide 3 0.10 65

Peptide 1 0.20 33


ED50: Defined in Table 6.

% Recovery of Activity: Calculated as described in Table 6.




70


peptide 1 was less active but did account for 33% of the

original activity.













CHAPTER VIII
DISCUSSION



It is clear that the substance migrating electrophore-

tically as peptide 2 contains the bulk of the biological

activity originally present in preparation I. It is anionic

at pH 4.8 and is readily digested by aminopeptidase.

Peptide 1, although less active and more resistant to the

action of aminopeptidase, does have significant stimulatory

activity. Whether these two peptides are structurally re-

lated remains to be established.

It is clear that future experimentation involves the

isolation of larger quantities of peptides 1 and 2 either

by electrophoresis or by column chromatography in order to

establish the following: 1) homogeneity, 2) biological

activity on a weight basis, and 3) the amino acid sequence.












BIBLIOGRAPHY


1. Hoppe-Seyler, F. A. 1933. Uber das Homarine, eine
bisher unbakannte tierische Base. Z. physiol. Chem.,
Hoppe-Seyler's. 22:105-155.

2. Beers, J. R. 1967. The species distribution of some
naturally-occuring quanternary ammonium compounds.
Comp. Biochem. Physiol. 21:11-21.

3. Gasteiger, E. L., P. C. Haake, and J. A. Gergen. 1960.
An investigation of the distribution and function of
homarine (n-methyl picolinic acid). Annals N. Y.
Acad. Sci. 90:622-636.

4. Levy, R. A. 1967. The independence of homarine from
osmoregulatory mechanisms in the ventral nerve cord of
Limulus polyphemus L. Comp. Biochem. Physiol.
23:631-644.

5. Leonard G. J. and K. MacDonald. 1963. Homarine
(n-methyl picolinic acid) in muscles of some Australian
crustacea. Nature. 200:78.

6. Dall, W. 1971. The role of homarine in decapod
crustacea. Biochem. Physiol. 39B:31-44.

7. Keyl, M. G., I. A. Michaelson, and V. P. Whittaker.
1957. Physiologically active choline esters in certain
marine gastropods and other invertebrates. J. Physiol.
139:434-454.

8. Welsh, T. H. and P. B. Prock. 1958. Quaternary
ammonium bases in coelenterates. Biol. Bull.
115(3):551-561.

9. Kravitz, E. A., S. W. Kuffler, D. D. Potler, and
N. M. van Gelder. 1963. Gama-aminobutvric acid and
other blocking compounds in crustacea. II. Peripheral
nervous system. J. of Neurophys. 26:729-738.

10. Haake, P. and J. Mantecon. 1964. Kinetic studies of
the decarboxylation of some n-substituted pyridine-
carboxylic acids. JACS. 86(23):5230-5234.








11. Green, R. W. and H. K. Tong. 1956. The constitution
of the pyridine monocarboxylic acids in their iso-
electric forms. JACS. 78:4896-4900.

12. Newman, F. M. 1973. Sample preparation procedures for
liquid scintillation counting. Biomedical Technical
Report. TR-551.

13. Cowey, C. B. and J. R. M. Forster. 1971. The essential
amino-acid requirements of the prawn Palaemon serratus.
The growth of prawns on diets containing proteins of
different amino-acid compositions. Marine Biology.
10:77-81.

14. Leete, E. 1965. Biosynthesis of alkaloids. Science.
47:1000-1006.

15. Ramstad E. and S. Agurell. 1964. Alkaloid bio-
synthesis. Ann. Rev. of Plant Physiology. 15:143-168.

16. Griffith, T. G., K. P. Hellman, and R. U. Byerrum.
1962.- Studies on the biosynthesis of the pyridine ring
of nicotine. Biochemistry. 1:336-340.

17. Lindstedt, K. J. 1971. Chemical control of feeding
behavior. Comp. Biochem. Physiol. 39:553-581.

18. Hodgson, E. S. 1955. Problems in invertebrate chemo-
reception. Quart. Rev. Biol. 30:331-347.

19. Kohn, A. J. 1961. Chemoreception in gastropod
molluscs. Am. Zool. 1:291-308.

20. Carr, W. E. S. 1967. Chemoreception in the mud snail,
Nassarius obsoletus. I. Properties of stimulatory
substances extracted from shrimp. Biol. Bull.
133:90-105.

21. Carr, W. E. S. 1967. Chemoreception in the mud snail,
Nassarius obsoletus. II. Identification of stimula-
tory substances. Biol. Bull. 133:106-127.

22. Gurin, S. and W. E. S. Carr. 1971. Chemoreception in
Nassarius obsoletus: the role of specific stimulatory
proteins. Science. 174:293-295.

23. Carr, W. E. S.. E. R. Hall, and S. Gurin. 1974.
Chemoreception and the role of proteins: a comparative
study. Comp. Biochem. Physiol. 47A:559-566.

24. Mangum, C. P. and C. D. Cox. 1971. Analysis of the
feeding response in the onuphid polychaete Diopatra
cuprea (Bosc.). Biol. Bull. 140:215-229.




74



25. Lowry, O. H., N. J. Rosebrough, A. L. Farr, and
R. L. Randall. 1951. Protein measurement with the
Folin phenol reagent. J. B. C. 193:265-275.












BIOGRAPHICAL SKETCH


Elizabeth Ruth Hall was born May 15, 1947 in Wharton,

Texas. At the age of two she moved to Corpus Christi, Texas

where she grew up and attended public schools.

In January, 1965 she graduated from W. B. Ray High

School. The rest of that school year was spent in attendance

at Del Mar Jr. College in Corpus Christi.

In September, 1965 she entered Texas Woman's University,

Denton, Texas, where she received her B. S. in biology in

1968 and her M. S. in zoology in 1969. While attending

Texas Woman's University, she held part-time employment as

a laboratory assistant for Dr. E. W. Hupp and as a teaching

assistant in various courses including mammalian physiology,

invertebrate zoology, comparative physiology, and

histotechniques.

Ms. Hall entered the Department of Biochemistry at

the University of Florida in July, 1968.








I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.




Samuel Gurin, Chairman
Professor of Biochemistry


I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.




William Carr
Associate Professor of Zoology


I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.




Eugey Sander
Asso iate Professor of Biochemistry








I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.




John Zoltewicz
Professor of Chemistry



This dissertation was submitted to the Graduate Faculty of
the Department of Biochemistry in the College of Arts and
Sciences and to the Graduate Council, and was accepted as
partial fulfillment of the requirements for the degree of
Doctor of Philosophy.

June, 1974


Dean, Graduate School




University of Florida Home Page
© 2004 - 2010 University of Florida George A. Smathers Libraries.
All rights reserved.

Acceptable Use, Copyright, and Disclaimer Statement
Last updated October 10, 2010 - - mvs