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 FULFILMENT 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. 11 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 C -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 endogenous ly 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 C -^-labeled compounds. Following injection of d,l tryptophan (benzene ring-C^-^(U)) , no cl^-homarine was found, thereby showing that tryptophan is not a major pre- cursor of homarine. Injection of acetic acid-2-C-'-^ did result in the production of C^-homarine . Previous investi- gators have shown that tryptophan is not labeled after the administration of C^-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-Cl4 acid. The homarine isolated had a VI 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 C -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- C^ *) into P. duorarum resulted in C -^-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 molecule (s) was estimated by ammonium sulfate precipitation, ultrafiltration through an Amicon UM 2 membrane, and Sephadex gel chromatography. The results obtained indicate that the active molecule(s) has a low molecular weight of approximately 1000. The activity of the major stimulant (s) was decreased 707o by aminopeptidase digestion, suggesting the involvement of a peptide. The Vll active molecule (s) 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 Hornarine (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 hornarine distribution; but they have yielded little enlightenment concerning its function, biosynthesis, or catabolism. The distribution of hornarine 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 hornarine in the shrimp, Palaemonetes vulgaris , to be 0.60 - 1.19 mg/gm of wet weight; yet, no trace of hornarine 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 hornarine 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/gra 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 0.033 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 10' 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 ai ciivity 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 CO2 . 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 juCi of C^- 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, C^-homarine was assumed to have been synthesized from C1 ^-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 endogenous ly 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 chromatography (anion and cation) , thin- layer chromatography, and Sephadex gel chromatography. Anion exchange chromatography. At a pH 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 AGl-x8 resin (0H~ form, 200-400 mesh) that was equilibrated and eluted with 0.5% NH,0H (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 10 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 + 11 of approximately 15 bed volumes of 0 . IN 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 x^ashed with 200-300 ml of water and eluted with 0 . IN 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 . IN 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 (raw 2 x 10°) 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 o CO tcj CD X X i !s u CD CD 'O g « g ttf V< V4 O Cu O co 0) O K U Cfl 4-1 O CD ih cd c oj -C CVH M • 4-1 CL) /— s d CD d CD CD H •HX-HX d CD U 4-1 g 4J •H 4J cd M c+-i g Xi^O 4J 03 03 O 4-) CO g X -H 4-J d O >^ [5 Cfl -H ,jG i-H <-H 03 d O •-n T3 fcO U O ■ d cu co O rH d E 4-> iHCU o 3 ox) Em •r4 i-l CD CD x ^ J_) O r-l 4-1 03 >-! 2: 3 OHU E oh .-1 O O > O O (!) OH M Cu CD CD 0) d co 43J3«W d^ c d 3 03 O CD O rH CD ,0 1)0 }-i -H CD 4-) 5-< CD r-l 0 4-) Oj O 4J 1 bO 03 CD 3 CO 0 O •H O X r-l JOi r-i O .ft, -^H CD 03 CD CO 13 rfaifc^ 33Nv<3^0S> CD QJ J5 42 42 m M 4-1 CU cd ^~N piw c r-l i O ■H d 6 d M o r-l C d o- O o E s~*. CN CJ •r-l oo i 4J ,doco a d r-< r-i 42 r-l o •^s 4-1 a •H X 4-1 r-l E QJ 0) CM O 42 42 ^ r-l 4-> 4-> r^ Cd M-4 R i CU CO ^.^ P-, d 4-J CO Co d d r^ CD , fn to CJ 4-J Pl, 42 15 ^ vl z aoNv^^os^v ah — 1 p. D. C B B 0 o H •r-i H CO M 5-1 g 4-> CO '.1 ,C! § 3 i co CO 3 i-l VD rH CU CN B b O ^v o o O CU M n ^CN M-l IH O •U t-l 14 X) T> 1 CO 'cd cu 0) e> ■M o 4-' •u C CM cfl Cli X CU r-l r-4 CJ CO CU 0 O X) cu i— i CO CO cd U •H •iH •H,c! !XrC Cl, QJ »> CU 0) CU U C C co - •H •H •~n a U U CT3 rH a cd c3 0 p1 $= 0 a rH b o O c> o^ rd c CM o M-l M-l m r-» CU o O n r-l ,£ ■U ^^ 4-J a d X o o cu r-i f- •H •H Q O 4.) 4J ^ r-l 3 03 CD Cd M-l rH U 3 CU cu tfl r-i P-i a PhPQ CtJ a CU Mjd CO 0 4-1 ■U o /~ V X J-i B cu CO M-l o n 4-J in C 4-J CO cu a 0) o en P co 5-i ctf r-l CU 3 H X PQ r-l W)4-> P. •H X r-l M-l CU Pn CU o r4 17 ^01 viz iDNvg^osgv ah 18 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 (250u) 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 oc-naphthol (equal volumes of 5N NaOH and 170 oi-naphthol in ethanol) as described by Leonard and MacDonald (5) . No ninhydrin-reacting compounds were detected on these chromatograms . 19 TABLE 2 Rf VALUES OF HOMARINE AND RELATED COMPOUNDS RUN ON MICROCRYSTALLINE CELLULOSE PLATES Solvent System Compound Rf I 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 butanol : acetic acid:water bSolvent System II: 95:5:5 methanol : acetic acid: water 20 Precipitation of Homarine with Ph o s phot imp; s tic Acid Homarine phosphotungstate, a relatively insoluble salt, was precipitated cold from an acidic homarine solution (approximately IN H„S0,) 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 dissolveu 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 2M H SO to a pH of 1 leaving a solution of homarine 2 4 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 21 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% NH4OH (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 HC1 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 ^min 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. 23 Shrimp Muscle blend in cold 95% CH3OH "1 Insoluble Soluble | dry, * \ Insoluble Soluble extract with 1^0 1 shake with CHCl. CHC1„ phase H20 phase apply to AGl-x8 column wash with 0.5% NH40H i \ Retained Unretarded I apply to AG50W-x8 wash with H2O \ * Retained Unretarded elute with 0 . IN HC1 i { Retained Eluted phosphotungstate ppt. Precipitate Supernatant dissolve 0.5N NaOH add 10% BaOH, 2N H.SO. 2 4 I \ Precipitate Homarine 24 Procedures Used in the Treatment of Radioactive Homarine Fractions volume were counted in a Beckman LS 230 liquid scintillation counter using a toluene based cocktail with 107o 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 500/u 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. 25 26 TABLE 3 HOMARINE CONCENTRATIONS FOUND IN CRAYFISH-FED SHRIMP Homarine Concentration (mg/gm) No. of Days Shrimp Shrimp Shrimp Average on Crayfish Diet 12 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 27 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 C*-^- 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 100°C 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 M c I-l 0) •H 03 o ^ M-4 •H C 03 o U CU cd o (D^rQ a g^2 4J H m CXr-4 rt o 03 0 CO c ,£) 0) cd ri -d- cd 0^ r~> £i CO i-H O cm U CO O o O a CO > co i—l c C a) :d ,•: o •H to cu CO •i-i 1 c co 4J 4-1 i-4 cd ■U O" C CJ OX •H r^ 0) cd X o CN CO U o X-d (U <4-4 o Q) C > U 1-4 cd D a cu cd G CU rH O co 0) M X -d •H p x 3 a; -U O H C-1 N cci •H O o ^ O 4-1 ■H co i-l CJ 4-) i O 00 03 a> 3--^ ^l X !-J P i-l o T3 1 m 3 CU X ^ r-l o X O S O CU CJ LO •H > C 1—1 M-i O B •H cd O < a> M o 4-) 03 CU a c cd E^ o a) c. 3 o 4-) •H ■H .-1 ,: : ■U C 0) c Cfl O T3 M-l •1-1 id CU cu O o cu -u ,r; -a •i-4 rH o 4-J c C 4-J O 0) o 3 o CO i—l 4J •H o cd 3.-I CO to IM !-i 6 o c 0) fe o •H M CU CX 03 c E CO oJ3 CU •H •H cd 03 X H r^ !-4 £ 4-J cd X T3 B 0) CO CU cu c o M ■u 4J ■H r\ s B cd ■U X M o p c ■U ai •H J-i ■ — I < — f •HX fe 14-1 CU a? 4-1 29 o I — O -r to *C UJ u. u. riwiVLZ 3ONV<320S<3V Af) 30 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 hydro lyzed, 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 C-1- -Labeled Compounds Additional evidence for the synthesis of homarine by shrimp was obtained by injecting C^- labeled compounds into the shrimp either intravenously or intramuscularly with the c o •H ■u o QJ Crj t-< fcJD o a i c r-l -H o « 0) 4-J O o I £t QJ QJ C 14-J •H O U 03 60 u-! B E o o O QJ • •rl £%<4-l C i-H •H O bO U u £ Cd T3 •H H >> C o x •H X cd CD 4-> m X C o ■u o o M <4-i 1 i o 0) C 6 cd 0 CO }-< XI c cd >H D- OJ CtJ 0) X 4J en H o o o •r4 ^Ht) CL, Cd a crt •H cd H )-l M cd I-l o 4-1 4-> « ,M cd s cd H CD o 0) Ci U r-i XX CO CJ 3 i-4 cd OT) co M M Pi H K O co < M T) B M-4 CU \o CC e m a cu CUM ctf c ^ 4-1 h •H ~^s 0 bO M <; O rt W 4J E c rl o o o E 1-^ •H QJ 0 440 P U O H co ,d CU u O M < E o fi cu -r) a ^ CO 13 CU M rt o H H CJ ^_^ o (!) K C* g 1) a. cu U a) H > cti o 4-1 CJ O CU H Pi •H O 3 -a C o a, E o o CU r-H CU X! CJ C fcO o 4J 0) &. e >> CU U N h a I CU 13 vO CO 00 CT* •H 4J CJ , ,£) CO H 4J CU C 4J CU CM > rt i-H o co •H }-4 4-i a) CJ -U rt m u & I4_| .. CU -H C CJ r-< o cm C o ^ 4-1 >.d 4-> & •H >o •H CN 4-1 •• CJ O rt CN CJ O •H ^O CM •H rt o cu c cu-h co co 13 CU CU 4J 4-1 ^ B cu 'H a, •M 4-1 4-1 CU CO CO CU O iM CU 3 X<-i H r-l CU o M C Q O CM o o •H CO 4-1 CJ U H M CJ rt rt E o o .C -M M CU CU rC a 4J rt CM M O O c >. rt M^ •M M > CU •h e 4-J a in rt •• m CJ ■• •h in CM C^ •M CJ <^ cu Cu C CO -H X) CO cu cu CO cu cu CO o CU i-l x d H M iM 0) •• o M M C CJ O 38 shrimp to form homarine was investigated by iniecting 15uCi of quinolinic-6-C 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. C-*- -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 C -trypto- phan made this pathway an attractive possibility. One shrimp was injected with 25uCi of 1-aspartic acid-C (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 C -aspartate can contribute carbon atoms to homarine, yet not as readily as C -acetate. 39 -7C-OH C-OH Quinolinic Acid C-OH Picolinic Acid Homarine Figure 10. A proposed pathway for the incorporation of carbon 6 of quinolinic acid into the homarine molecule. 40 o 3" H-C "CHOH P-O-CH2 Glycer aldehyde 3-Phosphate + 2CH2-rCOOH 'CH-'COOH Aspartic Acid /^COOH 1,2 dicarboxy-3, 4 hydroxy-piperidine COOH Quinolinic Acid Figure 11. Biogenetic scheme for the formation of quinolinic acid in higher plants and some microorganisms as proDOsed by Leete (14) and others (15, 16). 41 C -methionine . In order to determine whether the methyl group of homarine is derived from methionine, two shrimp were given intramuscular injections of 50uCi of 1-methionine (methyl- C) . The shrimp were homogenized after 12 hrs and their homarine extracted. The 3.2 mg of homarine isolated contained a total of 557 dpm. Aliquot s 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 C1^ 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 endogenous ly 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 C-"- - 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 C-1- -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 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. There are very few metabolic pathways known to give rise to quinolinic acid. Mention has been made (14-16) of 42 43 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 46 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. lO-^) . 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 e_t 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 47 sulfate precipitable, rton-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 48 cd CO W Q H H PL, w Pw w J23 H CJ rJ O o >< H H > H H U «J S3 o H O a CO CD Pd 0) cd ° 5 5-1 cd i-J -u a txo cd -H r-H CD iS CO o CO o o CN O CM O O r-4 CO o CM CM CM Ch v£3 CO CO i >^ rA M C Ml CO CO 4J X! rH CO a 4-1 0) i— t to 1-1 >* O >■, CJ cd pCi >^ w o CJ >^ i—i o CD c •H o >> r-H to •rl H CD C ■H CJ i-l to -U CD H CD a •H CJ >s rH M di ■u C a) Pw CD C •H CJ >-, H bO cd a) S3 03 r^ XI H >•, U cd CJ 13 C cd «w IH O e cd O >> a C cd S3 >^ CD CJ O 13 r-l o cd T) 13 CD X3 cd S3 o o S3 CM cd 53 M PJ •H 13 "0 cd 13 C cd M P3 •H cd H CD 4J cd IS cd CD 3 CO 0) cd s 49 by William Carr, Samuel Gurin, and Elizabeth Hall to isolate and characterize the major response-inducing molecule (s) 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 molecule(s) 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 ul of solu- tion per ml of sea water necessary to induce the proboscis search reaction in 50% of the test animals (effective dose for 507o of test animals = EDj-r,) • 50 51 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 7°C. The resulting 90% ammonium sulfate solution was then centrifuged for 30 min at 10,000 rpm at 0°C. 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 factor (s) in shrimp extracts is non-protein in character or is of low molecular weight. Similar experiments were run yielding comparable results. Ultraf i ltration Another portion of the prepared shrimp extract (38 ml) was ultraf iltered through an Amicon UM 2 membrane at 4 C 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 577o of the original activity, and the ultrafiltrate, with 35% of the original activity. 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