The Properties and Functions of Alanopine Dehydrogenase and Octopine Dehydrogenase from the Pedal Retractor Muscle of Strombida'e (Class Gastropoda)!

J. BALOWIN 2 and W. R. ENGLAN0 2

ABSTRACT: The pedal retractor muscles ofStrombidae contain high activities ofboth alanopine dehydrogenase and octopine dehydrogenase, raising questions as to the functions of these two enzymes during muscle anoxia associated with locomotion. Alanopine dehydrogenase and octopine dehydrogenase were iso lated from the pedal retractor muscle ofStrombus luhuanus, and their structural and kinetic properties investigated. Alanopine dehydrogenase occurs as a single electrophoretic form with a molecular weight of approx. 42,000. Octopine de hydrogenase was electrophoretically polymorphic, existing as three alleles in the population of animals studied. The major form of the enzyme had a molecular weight of approx. 39,000. Both enzymes displayed similar pH optima for the

forward (pyruvate reduction) reaction and similar Km values for the common substrates pyruvate and NADH. During bursts of leaping, both octopine and strombine/alanopine accumu lated in the pedal retractor muscles of Strombidae. However, during recovery from exercise, only strombine/alanopine accumulated. Octopine was a potent inhibitor ofthe forward reaction catalyzed by octopine dehydrogenase, and may act to prevent further octopine production during the recovery phase. The results of this study show that both alanopine dehydrogenase and octopine dehydro genase are functioning to catalyze the terminal step of anaerobic glycolysis during muscle anoxia associated with locomotion.

DURING SHORT-TERM BURSTS of anaerobic could compete for a common pool of pyru muscle work associated with locomotion, vate and NADH. The enzymes are (1) lactate adenosine 5'-triphosphate (ATP) may be dehydrogenase (pyruvate + NADH + H+ ~ obtained from substrate-level phosphoryla lactate + NAD+); (2) octopine dehydroge tions coupled to the glycolytic degradation nase (arginine + pyruvate + NADH + H+ ~octopine of carbohydrates. The final step of this path + NAD+ + H 2 0); (3) alanopine way serves to reoxidize NADH produced by dehydrogenase (alanine/glycine + pyruvate the glyceraldehyde phosphate dehydrogenase + NADH + H+ ~ alanopine/strombine + reaction, with pyruvate functioning as the ter NAD+ + H 2 0). minal electron acceptor. In mollusk muscle, It is generally held that competition be this pyruvate reduction may be catalyzed by tween lactate dehydrogenase and octopine de at least three enzymes, which potentially hydrogenase is avoided by the presence in the muscle of much greater activities of one of

I This research was conducted as part of the Alpha these enzymes, or by kinetic and regulatory Helix Cephalopod Expedition to the Republic of the properties that result in the two reactions pro Philippines, supported by National Science Foundation ceeding in opposite directions during aerobic grant PCM 77-16269 to J. Arnold. This study was also -anaerobic transitions (Baldwin and Opie supported in part by the Australian Research Grants Committee. 1978, Fields et al. I976b, Regnouf and 2 Monash University, Department of Zoology, van Thoai 1970, Storey 1977, Zammit and Clayton, Victoria 3168, Australia. Newsholme 1976). However, in at least some 381

, , ,_ "'.. ,,, "" > " ,", • '-'~ .. < I ,. <. ". ,n "",."" " .'. T' .'- , ,- " r" ".. " ..-,.-... 'f' 382 PACIFIC SCIENCE, Volume 36, July 1982 gastropods and bivalves, both octopine and assaying alanopine dehydrogenase, octopine lactate are produced within the same muscle, dehydrogenase, lactate dehydrogenase, and although during different physiological states phosphofructokinase. All assays were com associated with muscle anoxia (Baldwin, Lee, pleted within 90 min of tissue preparation. and England 1981; Gade 1980). Enzyme activities were measured with a Less information is available on the distri Vnicam SPI800 orZeiss DM4 recording spec bution, properties, and biological functions of trophotometer, and cell temperatures were alanopine dehydrogenase and possible com controlled with a circulating water bath. petition between this enzyme and octopine Citrate synthetase was assayed at 412 nm, and and lactate dehydrogenases in mollusk mus the other enzyme reactions were followed by cles used in powering locomotion (Fields 1976; absorbance changes at 340 nm due to oxida Fields and Hochachka 1981; Fields et a!. 1980; tion of NADH or reduction of NADP. Suit de Zwaan, Thompson, and Livingstone 1980). able controls were run to allow for nonspecific Isolation and identification of the product activity, and all determinations were made in strombine from the gastropod Strombus gigas triplicate at 25°C, which approximates the (Sangster, Thomas, and Tingling 1975) led us habitat temperature ofthe animals examined. to examine the distribution of the three de Assays were carried out with 2-25 J.lI of hydrogenases in pedal retractor muscles of a muscle extract in a total reaction volume of I range of gastropods. m!. The composition ofthe reaction mixtures, selected to give maximum activities, were as follows: (1) octopine dehydrogenase: 5 mM sodium pyruvate, 20 mM arginine, 0.2 mM MATERIALS AND METHODS NADH, 50 mM sodium phosphate buffer, pH 7.0; (2) lactate dehydrogenase: 2.5 mM Experimental Animals sodium pyruvate, 0.2 mM NADH, 50 mM Gastropods were collected on the Great sodium phosphate buffer, pH 7.4; (3) alano Barrier Reef, and the Victorian Coast, Aus pine dehydrogenase: 5 mM sodium pyruvate, tralia, and in the Tanon Strait, Philippine 200 mM glycine, 0.2 mM NADH, 50 mM Islands, during the R/V Alpha Helix expe sodium phosphate buffer, pH 7.0; (4) a dition. Animals used for muscle metabolite glycerophosphate dehydrogenase: 0.5 mM studies were held for up to 5 days at 23°C in dihydroxyacetone phosphate, 0.2 mM NADH, outdoor circulating seawater tanks. 50 mM sodium phosphate buffer, pH 7.4; (5) phosphofructokinase: 0.1 mM NADH, 0.3 mM sodium cyanide, 6 mM fructose-6 Determination afthe Maximum Activities of Enzymes in Pedal Retractor Muscles phosphate, 2 mM AMP, I mM ATP, 5 mM magnesium chloride, 100 mM potassium chlo Fresh pedal retractor muscles were cut into ride, 2 IV triosephosphate isomerase, 2 IV small pieces and homogenized with 10 vol aldolase, 2 IVa-glycerophosphate dehydro of ice-cold buffer [50 mM sodium phosphate, genase, 50 mM Tris-HCI buffer, pH 8.2; (6) 10-4 M ethylenediaminetetraacetic acid hexokinase: 0.4 mM NADP, 7.5 mM mag (EDTA), 10-4 M 1,4-dithioerythritol (DTE), nesium chloride, 1 mM EDTA, 5 mM 2 pH 7.5]. Triton X-IOQ (1% vol/vol) was added mercaptoethanol, 1.5 mM potassium chlo to an aliquot of the homogenate, which was ride, 2.5 mM ATP, 1.5 mM glucose, 10 mM then incubated on ice for 30 min, followed creatine phosphate, 2 IV glucose-6-phosphate by centrifugation at 600 x g to remove cell dehydrogenase, 2 IV creatinekinase, 50 mM debris. This supernatant was used for assay Tris-HCl buffer, pH 7.5; (7) glutamate ing hexokinase, glutamate oxaloacetate trans oxaloacetate transaminase: 0.2 mM NADH, aminase, a-glycerophosphate dehydrogenase, 10 mM a-ketoglutarate, 20 mM aspartate, and citrate synthetase. The remaining portion 0.1 mM pyridoxal-5-phosphate, 2 IV NAD of the original homogenate was centrifuged malate dehydrogenase, 50 mM Tris-HCI as above, and the supernatant was used for buffer, pH 7.5; (8) citrate synthetase: 0.5 mM Dehydrogenases in Strombidae-BALDwlN AND ENGLAND 383

oxaloacetate, 0.1 mM acetyl CoA, 0.2 mM Fractions with the highest specific activity 5,5'-dithiobis-(2-nitrobenzoic acid), 50 mM were stored at 4°C in 80% saturated am Tris-HCI buffer, pH 8.0. monium sulfate. The rate measured with the octopine de The portion ofthe original supernatant pre hydrogenase and alanopine dehydrogenase cipitating between 55 and 90% ammonium reaction mixtures includes lactate dehydro sulfate saturation was used for the isolation of genase activity; thus, values were corrected by alanopine dehydrogenase. This precipitate was subtracting the rate due to lactate dehydro subjected to CM-cellulose chromatography as genase alone when arginine or glycine was described for octopine dehydrogenase. Pooled omitted from the assay. fractions from the CM-cellulose column were concentrated by ammonium sulfate precipita Isolation ofAlanopine Dehydrogenase and tion and dialyzed against 25 mM Tris-HCI Octopine Dehydrogenase from the Pedal buffer, pH 8.0. The dialyzed sample was loaded onto a DEAE-cellulose column equili Retractor Muscle ofStrombus luhuanus brated with the dialysis buffer, and the enzyme All steps in the isolation procedures were was eluted as a single peak of activity with a carried out at 0-4°C; buffers contained 10-4 linear gradient from 0 to 300 mM sodium M EDTA and 10-4 M DTE. Pedal retractor chloride in the column equilibration buffer. muscles were homogenized in 5 vol of 50 mM Pooled fractions were concentrated by mem sodium phosphate buffer, pH 7.0, using a brane filtration and further purified by gel Sorvall omni-mixer. The homogenate was filtration as described for octopine dehydro centrifuged at 10,000 x g for 30 min, and the genase. Fractions with the highest specific pellet discarded. activities were stored at 4°C in 80% saturated The portion of the supernatant precipita ammonium sulfate. ting between 0 and 55% ammonium sulfate saturation was collected by centrifugation and Determination ofMolecular Weight used for the isolation of octopine dehydro The molecular weights ofStrombus luhuanus genase. The precipitate was dissolved in a alanopine dehydrogenase and octopine dehy small volume of 10 mM sodium phosphate drogenase were estimated by gel filtration on buffer, pH 6.5, and dialyzed against a large volume of the same buffer. The dialyzed Sephadex G 100. The column (2.5 x 85 cm) sample was applied to a O-(carboxymethyl) was run at 4°C, with 50 mM Tris-HCI buffer, pH 7.6, containing 100 mM sodium chloride. (CM) cellulose column (Whatman CM23) It was calibrated with ribonuclease A (13,700 equilibrated with the dialysis buffer, and octo mol wt), chymotrypsinogen A (25,000 mol pine dehydrogenase activity was eluted by wt), ovalbumin (43,000 mol wt), and bovine washing with this buffer. Pooled fractions serum albumin (67,000 mol wt). The void from the CM-cellulose column were concen volume was determined with Blue Dextran trated by ammonium sulfate precipitation 2000 (Pharmacia gel filtration kit, low molec and dialyzed against 25 mM Tris-acetate ular weight). Samples were applied in 5 ml of buffer, pH 8.4. The dialyzed sample was buffer, and 5-ml fractions were collected at a loaded onto a O-(diethylaminoethyl) (DEAE) flow rate of 30 mllhr. Molecular weights were cellulose column (Whatman DE22) equili calculated from the plot of Vel V versus brated with the dialysis buffer. Octopine de o log molecular weight, where V is the elution hydrogenase activity was eluted as up to three e volume of the protein and V is the void separate peaks by using a linear gradient from o volume of the column. oto 250 mM sodium chloride in the column equilibration buffer. The peaks were concen Protein Determinations trated separately by membrane filtration (Amicon, PMlO) and further purified by gel The protein concentrations of samples as filtration on a Sephadex G 100 column equili sayed during enzyme purification were deter brated with 50 mM Tris-HCI buffer, pH 7.6. mined by ultraviolet (UV) absorption using

" , ". " r """~ "'rO~ "..... , "'" ""~, "_,,, ~'U'~ ",.,...",r ,.. """, ". 384 PACIFIC SCIENCE, Volume 36, July 1982 the following relationship: protein concentra were determined with an amino acid analyzer tion (mg/ml) = 1.55A28o - 0.75A260 (Layne after the perchloric acid extracts were dried 1957). under vacuum and dissolved in an appro priate buffer. Electrophoresis Exercising Animals Alanopine dehydrogenase and octopine de hydrogenase preparations were run on cel Strombus luhuanus, S. gibberulus, and lulose acetate gels (Cellogel, Chemtron, Lambis lambis were exercised in small tanks of Milano) using 100 mM Tris-citrate buffer, seawater. Leaping behavior was initiated by pH 7.5. The gels were stained for activity as adding crushed specimens ofpredatory Conus described by Fields et al. (1976b), using strom spp. to the water. The animals responded bine, alanopine, or octopine as substrates. within several minutes by violently leaping Proteins were stained with Coomassie blue. about the tank using the pedal retractor muscle for propulsion. This activity continued Determination ofMuscle Metabolites at up to 16 leaps/min for 5-10 min, after which the animals withdrew into the shell and Entire pedal retractor muscles were rapidly failed to respond when further attempts were removed and frozen in liquid nitrogen. The made to elicit activity. Individuals used for frozen tissue was crushed in a percussion recovery experiments were removed from the mortar, and then ground to a fine powder exercise tank, washed free of mucus, and under liquid nitrogen with a mortar and placed in fresh aerated seawater, or in air, for pestle. The muscle powder was extracted with the required time. 10 vol of ice-cold 0.6 M perchloric acid in a Sorvall omni-mixer. The perchloric acid ex tracts were centrifuged at 10,000 x g for 20 min at 4°C, and the supernatants were neutra RESULTS lized with 5 M potassium carbonate. After Enzyme Activities in Gastropod Pedal standing on ice for several hours, precipitated Retractor Muscles potassium perchlorate was removed by centri fugation. The samples were stored at - 20°C The maximum activities of alanopine de until assayed. hydrogenase, octopine dehydrogenase, and Octopine was determined using the purified lactate dehydrogenase assayed in super Strombus luhuanus octopine dehydrogenase. natants of pedal retractor muscles from a The reaction mixture contained 100 mM Tris range of gastropods are shown in Table 1. HCl buffer, pH 9.0, 100 mM hydrazine, 10 Alanopine dehydrogenase was present in mM NAD+, and 10 IV octopine dehydro twelve of the eighteen families surveyed, with genase. Strombine and alanopine were as highest activities occurring together with high sayed with the same reaction mixture, but activities of octopine dehydrogenase in the using 10 IV alanopine dehydrogenase in place Strombidae. In the Conidae, Cypraeidae, ofoctopine dehydrogenase. This assay did not Cassidae, Cymatiidae, and Thaididae, signi distinguish between strombine and alanopine, ficant activities of alanopine dehydrogenase and the results are presented as the sum of occur in the absence of high activities of the two compounds (strombine/alanopine). octopine dehydrogenase and lactate dehydro Arginine was measured by the method of genase. Giide and Grieshaber (1975), and arginine On the basis of these results, the pedal re phosphate as described by Grieshaber and tractor muscle of Strombus luhuanus was Giide (1976). Pyruvate was measured en chosen for the purification of both alanopine zymatically using lactate dehydrogenase. dehydrogenase and octopine dehydrogenase. Adenylates were determined with test kits Strombus luhuanus, S. gibberulus, and Lambis (Boehringer Mannheim). Alanine and glycine lambis were used for metabolite studies be- Dehydrogenases in Strombidae-BALDwIN AND ENGLAND 385

TABLE 1 MAXIMUM ACfIVITIES OF ALANOPINE DEHYDROGENASE, OCfOPINE DEHYDROGENASE, AND LACfATE DEHYDROGENASE IN PEDAL RETRACfOR MUSCLES OF GASTROPODS

ALANOPINE OCfOPINE LACfATE DEHYDROGENASE DEHYDROGENASE DEHYDROGENASE

Strombidae, Strombus aurisdianae 163 410 13.8 S. canarium 240 241 12.1 S. gibberulus 90 702 13.0 S.labiatus 227 369 3.5 S. lentiginosus 269 442 25 S.luhuanus 230 585 5.4 S. sinuatus 153 143 12.3 Lambis lambis 139 606 15.2 L. mil/epeda 150 94 3.4 L. scorpius 55 266 9.8 Tibia martinii 61 111 6.8 Cymatiidae, Cymatium pi/eare 158 <0.2 2.7 Cypraeidae, Cypraea tigris 65 <0.2 0.7 Thaididae, Thais tuberosa 25.3 <0.2 <0.2 Conidae, Conus arenatus 19.5 <0.2 <0.2 Bursidae, Bursa sp. 15.6 5.2 4.5 Cassidae, Cypraecassius rufa 14.2 <0.2 1.4 Nassariidae, Nassarius coronatus 7.8 148 45 N. glansparticeps 4.5 87 21 Buccinidae, Cantharus undosus 4.4 64 3.9 Neritidae, Melanerita atramentosa 3.7 <0.2 1.2 Xenophoridae, Xenophora crispa 3.7 2.9 <0.2 Mitridae, Mitra eremitarum 2.3 <0.2 0.7 Muricidae, Dicathais texti/osa <0.2 40 1.8 Murex tribulus <0.2 14 <0.2 Patellidae, Cel/ana tramoserica <0.2 <0.2 <0.2 Siphonariidae, Siphonaria diemenensis <0.2 <0.2 2.4 Trochidae, Austrocochlea concamerata <0.2 <0.2 <0.2 Turbinidae, Turbo argyrostomus <0.2 <0.2 <0.2 Volutidae, Cymbiola vesperti/io <0.2 <0.2 24.8

NOTE: Data expressed as I'mole NADHjminjg wet wt muscle at 25°C.

TABLE 2 MAXIMUM ACTIVITIES OF ENZYMES IN PEDAL RETRACTOR MUSCLES OF Strombus luhuanus, Strombus gibberulus, AND Lambis lambis

ENZYME S.luhuanus S. gibberulus L. lambis

Hexokinase <0.1 < 0.1 <0.1 Phosphofructokinase 20.0 ± 1.7 14.5 ± 0.5 9.5 a-Glycerophosphate dehydrogenase 0.40±0.03 1.3 ± 0.07 <0.1 Glutamate oxaloacetate transaminase 7.6 ± 0.1 8.9 ± 1.5 Citrate synthetase 0.41 ±0.02 0.47± 0.04 0.20 A1anopine dehydrogenase 245 ±5 89 ±1O 140 Octopine dehydrogenase 576 ±9 651 ±51 615 Lactate dehydrogenase <0.1 < 0.1 <0.1

NOTE:~Data expressed asp-mole substratejmin/g wet wt muscle at 25°C. Values for S./uhuanus and S. gibberulus are means ± ranges for determinations on two individuals. Values for L. lambis are means for three determinations on one individual.

" ,, ~ .~, " I '" ,,,' ~ " .,-, r , , ,. _ ,., , ,• T" " " ,,_~,. _ _" "....,." 386 PACIFIC SCIENCE, Volume 36, July 1982

cause of their availability and the ease with gels were stained with MS-alanopjne or D which the animals could be exercised. The strombine. No band was observed when L maximum activities of a number of enzymes strombine was used as substrate. The final involved in aerobic and anaerobic energy alanopine dehydrogenase preparation was es metabolism in the pedal retractor muscles of timated to be about 70% homogenous on the S. luhuanus, S. gibberulus, and L. lambis are basis ofprotein and activity staining following shown in Table 2. The small amount oflactate electrophoresis. Ithad a specific activity of200 dehydrogenase activity in these muscle homo IV/mg protein, and was free of octopine de genates (Table 1) disappeared following di hydrogenase activity. alysis, and is assumed to be an artifact re Octopine dehydrogenase occurs as three sulting from the presence ofarginine, alanine, electrophoretically distinguishable anodally and particularly glycine in the tissue extracts migrating bands of enzyme activity in the (see Table 8) serving as substrates for octopine pedal retractor muscles ofStrombus luhuanus. dehydrogenase and alanopine dehydrogenase. Individual animals possess either one or two of these three bands. When purifying the Purification ofAlanopine Dehydrogenase and enzyme from pooled samples, the three bands Octopine Dehydrogenase were separated as nonoverlapping peaks of activity by DEAE-cellulose chromatography. Alanopine dehydrogenase was present as a The major peak, accounting for up to 80% of single anodal electrophoretic band in pedal the total octopine dehydrogenase a9tivity in retractor muscles of all Strombus luhuanus pooled samples ofpedal retractor muscle, was examined. This band migrated at about twice used for structural and kinetic studies and the rate of octopine dehydrogenase, and for metabolite determinations. The purified the two enzymes were readily separated enzyme gave single coincident protein and on DEAE-cellulose chromatography. Coin- activity bands on electrophoresis, had a spe cident bands of alanopine dehydrogenase cific activity of 780 IV/mg protein, and was activity were obtained when electrophoretic free ofalanopine dehydrogenase activity. TABLE 3

Sirombus luhuanus ALANOPINE DEHYDROGENASE: ApPARENT Km VALUES FOR SUBSTRATES

APPARENT Km SUBSTRATE COSUBSTRATE (mM)

Pyruvate 200 mM glycine, 0.2 mM NADH 3.1 10 mM glycine, 0.2 mM NADH 2.9 200 mM alanine, 0.2 mM NADH 2.8 10 mM alanine, 0.2 mM NADH 2.4 Glycine 5 mM pyruvate, 0.2 mM NADH 200 0.5 mM pyruvate, 0.2 mM NADH 200 Alanine 5 mM pyruvate, 0.2 mM NADH 90 0.5 mM pyruvate, 0.2 mM NADH 70 NADH 200 mM glycine, 5 mM pyruvate 0.04 10 mM glycine, 0.5 mM pyruvate 0.04 200 mM alanine, 5 mM pyruvate 0.06 10 mM alanine, 0.5 mM pyruvate 0.03 MS-Alanopine 5mM NAD+ 3.2 I mMNAD+ 3.2 D-Strombine 5mMNAD+ 2.8 I mMNAD+ 2.8 NAD+ 10 mM MS-alanopine 0.25 0.5 mM MS-alanopine 0.40 2 mM D-strombine 0.74

ASSAY CONDITIONS: forward reaction. 50 mM sodium phosphate buffer, pH 7.0; reverse reaction, 50 mM Tris-HCl buffer, pH 8.0. Assay temperature, 25°C. Apparent Km values were detennined from Lineweaver-Burk and Cornish-Bowden plots. Dehydrogenases in Strombidae-BALDwIN AND ENGLAND 387

Molecular Weight ofAlanopine TABLE 4 Dehydrogenase and Dctopine Dehydrogenase Strombus luhuanus ALANOPINE DEHYDROGENASE: INHIBITION CONSTANTS (K;) FOR THE FORWARD Gel filtration on Sephadex G 100 gave REACTION a molecular weight of 42,000 for Strombus luhuanus alanopine dehydrogenase. A value K; of 47 000 has been reported for the enzyme SUBSTRATE INHIBITOR (mM) from' oyster adductor muscle (Fields and Hochachka 1981). The molecular weight of Pyruvate Octopine 17.5 o-Strombine 2.8 39000 obtained for octopine dehydrogenase MS-Alanopine 8.5 co~responds to the single subunit enzyme o~ Glycine Octopine 18 bivalves (Baldwin and Opie 1978, Olomuckl o-Strombine 2.8 et al. 1972) and the enzymes from cephalopod Alanine Octopine 18 MS-Alanopine 8.5 mantle (Fields, Baldwin, and Hochachka NADH NAD+ 0.23 1976a). ASSAY CONDITIONS: 50 mM sodium phosphate buffer. pH 7.0. 25°C; K, values were determined from Dixon plots using three concentrations of Kinetic Properties ofStrombus luhuanus substrate and saturating concentrations ofcosubstrates. Alanopine Dehydrogenase

EFFECT OF pH ON ACTIVITY: The pH profiles similar to those obtained for octopine dehy at saturating substrate concentrations show drogenases of bivalves and cephalopods optima at about pH 7.0 for the forward (pyru (Baldwin and Opie 1978, Fields et al. 1976a), vate reduction) reaction and 8.5 for the re with a pH optimum of about 7.0 for the for verse reaction. The ratio of the maximum ward reaction and a broad peak at 9.4 for the activities at these pH optima is 6 : I in favor of reverse reaction. The relative activities under the forward reaction. optimal conditions were in the ratio 21 :I in SATURATION KINETICS: The enzyme dis favor of the forward reaction. played Michaelis-Menten saturation kinetics SATURATION KINETICS: Michaelis-Menten for all substrates of both the forward and saturation kinetics were obtained for all sub reverse reactions. Substrate inhibition did strates. Substrate inhibition occurred at not occur at high concentrations of pyruv~te, octopine concentrations above 5 mM, but alanine, glycine, NAD+ , NADH, D-strombIne, arginine, pyruvate, NAD+, and ~ADH were or MS-alanopine. No activity was observed not inhibitory at high concentratIOns. The Km when L-strombine was used as a substrate values for these substrates are listed in Table for the reverse reaction. The K (apparent m 5. As with other molluscan octopine dehydro Michaelis constant) values for these substrates genases (Fields et al. 1976a; Hochachka, are listed in Table 3 and can be compared with Hartline, and Fields 1977), the Km values for the substrate concentrations present in the pyruvate and arginine were influenced by the Strombus luhuanus pedal retractor muscle cosubstrate concentration (arginine or pyru (Table 8). vate). Increasing the concentration ofone sub The effectiveness ofthe products ofthe for strate decreased the K value for the other. ward reactions catalyzed by both alanopine m The K values listed in Table 6 show that dehydrogenase and octopine dehydrogenase octopine'was a potent inhibitor ofthe forward in inhibiting alanopine dehydrogenase a~e reaction, with K octopine abo~t one-twelfth given as K (inhibition constant) values In j j the highest octopine concentratIOn measured Table 4. in the Strombus luhuanus pedal retractor Kinetic Properties ofStrombus luhuanus muscle (Table 8). This product inhib.iti.on was Dctopine Dehydrogenase competitive with respect to both arginine and pyruvate. The effects ofphysiological con~en

EFFECT OF pH ON ACTIVITY: The pH profiles trations ofoctopine on the Km values obtained at saturating substrate concentrations were for these substrates are shown in Table 7.

"". , ~n, 'n"""" ,.,,~- < ,_.~" ".,,~, t «" " .. _, ~ ..,~ .~ ,,,,,.,.,, , , - 388 PACIFIC SCIENCE, Volume 36, July 1982

TABLE 5

Strombus luhuanus OcrOPINE DEHYDROGENASE: ApPARENT Km VALUES FOR SUBSTRATES

APPARENT K m SUBSTRATE COSUBSTRATE (mM)

Pyruvate 1 mM arginine, 0.1 mM NADH 5.0 10 mM arginine, 0.1 mM NADH 3.0 10 mM arginine, 0.05 mM NADH 3.2 Arginine 0.25 mM pyruvate, 0.1 mM NADH 20.0 0.5 mM pyruvate, 0.1 mM NADH 11.0 1 mM pyruvate, 0.1 mM NADH 6.0 1 mM pyruvate, 0.05 mM NADH 5.5 NADH 20 mM arginine, 5 mM pyruvate 0.06 1 mM arginine, 0.5 mM pyruvate 0.04 Octopine 0.1 mM NAD+ 0.18 0.5mMNAD+ 0.18 1 mMNAD+ 0.18 1 mM octopine 0.32 5 mM octopine 0.33

ASSAY CONDITIONS: forward reaction, 50 mM sodium phosphate buffer, pH 7.0; reverse reaction, 50 mM Tris-Hel buffer, pH 8.0. Assay temperature, 25°C. Apparent Km values were determined from Lineweaver-Burk and Cornish-Bowden plots.

TABLE 6 TABLE 7 Strombus luhuanus OCTOPINE DEHYDROGENASE: Strombus luhuanus OcrOPINE DEHYDROGENASE:

INHIBITION CONSTANTS (K;) FOR THE FORWARD ApPARENT K m VALUES FOR ARGININE AND PYRUVATE REACTION AT VARIOUS OCTOPINE CONCENTRATIONS

ASSAY CONDITIONS: 50 mM sodium phosphate buffer. pH 7.0, 25°C; Km ASSAY CONDtTtONS: 50 mM sodium phosphate buffer, pH 7.0, 25°C; K, arginine: I mM pyruvate, 0.2 mM NADH; Km pyruvate: 10 mM arginine, 0.2 values were determined from Dixon plots using three concentrations of mM NADH. Apparent Km values were determined from Lineweaver-Burk substrate and saturating concentrations ofcosubstrates. plols.

TABLE 8 MAXIMUM CONCENTRATIONS OF METABOLITES MEASURED IN PEDAL RETRACTOR MUSCLES OF Strombus luhuanus

CONCENTRATION SUBSTRATE STATE OF ANIMAL (jJmole/g wet wt muscle)

Pyruvate Resting in water < 0.2 Pyruvate Exercised < 0.2 Glycine Resting in water 147 Alanine Resting in water 5.2 Octopine Exercised 2.3 Alanopine/strombine Recovery in air 5.9 Arginine Resting in water 9.6 Arginine Exercised 10.8

, " •• ,,, "'" '" 'J ',r " , ,.,. n _,... , , • " Dehydrogenases in Strombidae-BALDwIN AND ENGLAND 389

TABLE 9

CONCENTRATIONS OF STROMBINE/ALANOPINE, OCTOPINE, AND ARGININE PHOSPHATE IN PEDAL RETRACTOR MUSCLES OF Strombus luhuanus, Strombus gibberulus, AND Lambis lambis AT REST AND IMMEDIATELY FOLLOWING EXERCISE

CONCENTRATION (Jlmole/g wet wt muscle)

MUSCLE STROMBINE/ ARGININE SPECIES STATE n ALANOPINE OCTOPINE PHOSPHATE

Strombus luhuanus Rest 5 0.5 0.5 11.5 (0.2-0.9) « 0.2-1.0) (10.1-12.4) 29 leaps I 1.6 1.2 10.0 33 leaps I 2.2 1.6 8.2 Strombus gibberulus Rest 6 0.3 0.7 10.2 « 0.2-0.8) « 0.2-1.3) (8.5-12.8) 108 leaps I 1.3 1.9 7.5 125 leaps I 2.9 2.5 6.5 Lambis lambis Rest 2 0.3 0.4 6.6 (0) (0.3-0.5) (6.2-6.9) 43 leaps 0.9 0.7 4.4 52 leaps 1.0 1.6 2.9

NOTE: n is the number of individual animals assayed. Values for resting animals are means, with ranges in parentheses.

Effects ofActivity on Pedal Retractor Muscle aminase occur in the mantle and fin muscles Metabolites of active pelagic squids that use aerobic car bohydrate catabolism during prolonged pe The effects ofexercise on the concentrations riods of rapid swimming (Baldwin, this issue; of strombine/alanopine, octopine, and ar Hochachka et al. 1975). Muscles that rely ginine phosphate in the pedal retractor mus heavily on anaerobic metabolism during short cles of Strombus luhuanus, S. gibberulus, and bursts of maximal activity and have little Lambis lambis (freeze-clamped immediately capacity in sustaining high levels of aerobic following activity) are shown in Table 9. work, show high activities of phosphofructo Concentrations of strombine/alanopine, kinase and octopine dehydrogenase, but lower octopine, arginine phosphate, and the energy activities of (X-glycerophosphate dehydro charge in pedal retractor muscles ofStrombus genase, glutamate oxaloacetate transaminase, luhuanus at rest, immediately following exer and Krebs cycle enzymes. Examples include cise, and after 10 min and 30 min recovery in the scallop fast adductor muscle and the water and in air are shown in Table 10. muscles of less active cephalopods (Alp, Newsholme, and Zammit 1976; Baldwin, DISCUSSION this issue; Baldwin and Opie 1978; Fields Enzyme Profiles in Pedal Retractor Muscles et al. 1976a; Zammit and Newsholme 1976). ofStrombidae Muscles with low maximum rates of both aerobic and anaerobic energy production Molluscan muscles used in powering loco have low activities of octopine dehydro motion differ markedly in the capacity to pro genase, phosphofructokinase, (X-glycerophos duce ATP under both aerobic and anaerobic phate dehydrogenase, glutamate oxaloacetate conditions. These differences are reflected in transaminase, and Krebs cycle enzymes (Alp the maximum activities ofassociated enzymes. et al. 1976, Baldwin and Opie 1978, Zammit High activities ofphosphofructokinase, citrate and Newsholme 1976). synthetase, and (X-glycerophosphate dehy The relatively high activities of octopine drogenase or glutamate oxaloacetate trans- dehydrogenase and phosphofructokinase,

, • _ • ' , ," , ,• ." , ~ .. , , ". " .. ~ ", ", ,, _. J • ". ., " , ,., - • ~ 390 PACIFIC SCIENCE, Volume 36, July 1982

TABLE 10

CONCENTRATIONS OF STROMBINE/ALANOPINE, OCTOPINE, ARGININE PHOSPHATE, AND ENERGY CHARGE IN PEDAL RETRACTOR MUSCLES OF Strombus luhuanus AT REST, IMMEDIATELY AFTER EXERCISE, AND DURING RECOVERY IN AIR AND IN WATER FOLLOWING EXERCISE

CONCENTRATION (~mole/g wet wt muscle)

STROMBINE/ ARGININE ENERGY MUSCLE STATE n ALANOPINE OCTOPINE PHOSPHATE CHARGE*

Resting in water 6 0.6 0.6 10.8 0.90 (0.2-1.0) ( <0.2-1.0) (7.4-12.4) (0.88-0.91) Exercised 6 1.2 1.5 9.9 0.90 (0.5-2.2) (0.8-2.3) (8.2-11.9) (0.89-0.91) 10 min recovery in water 4 1.2 1.2 9.5 0.90 (0.8-1.4) (0.8-2.1) (8.5-10.6) (0.89-0.91) 10 min recovery in air 4 1.4 1.1 8.0 0.86 (1.1-2.2) (0.4-1.9) (6.5-8.6) (0.83-0.89) 30 min recovery in water 4 2.2 1.4 9.2 0.87 (1.2-4.6) (1.1-1.8) (8.0-10.6) (0.82-0.90) 30 min recovery in air 4 3.7 1.9 7.1 0.84 (2.3-5.9) (1.3-2.2) (6.2-9.0) (0.79-0.89)

NOTE: n is the number ofindividual animals assayed. Values are means, with ranges in parentheses. • Energy charge was calculated according to Atkinson (1977) as ([ATPj + [ADPj/2)/([ATPJ + [ADPj + [AMP]). and low activities of hexokinase, a-glycero individual Strombus luhuanus pedal retractor phosphate dehydrogenase, glutamate oxaloa muscles suggests that the enzyme is polymor cetate transaminase, and citrate synthetase phic, existing as three alleles. If the enzyme in the pedal retractor muscles of Strombus is monomeric, as suggested by the molecular luhuanus, S. gibberulus, and Lambis lambis weight of 39,000, then individuals homo (Table 2) are indicative of muscles that rely zygous for anyone of the three alleles should on anaerobic ATP production during short give a single band ofoctopine dehydrogenase bursts of maximum activity, with octopine activity. Individuals heterozygous for any two accumulating as an end product of anaerobic of the three alleles should show two octopine glycolysis. dehydrogenase bands. As expected from this Alanopine dehydrogenase does not occur interpretation, no individuals possessing all at significant activities in the fast adductor three bands were observed. Similar genetic muscle ofthe scallop Placopeten magellanicus variation has been reported for octopine (de Zwaan et al. 1980), or in the mantle, fin, dehydrogenase in bivalve adductor muscle arm, funnel or retractor muscles of cepha (Beaumont, Day, and Giide 1980). lopods (Baldwin, this issue). The high acti vities of this enzyme in Strombus and Lambis How Many Enzymes Are Responsiblefor muscles used for powering short-term bursts Octopine, Strombine, and Alanopine of rapid leaping suggest that strombine Dehydrogenase Activity in Pedal Retractor or alanopine, in addition to octopine, may Muscles ofStrombidae? accumulate during muscle anoxia associated with locomotion in these animals. Octopine dehydrogenase and strombinej alanopine dehydrogenase activities in the Strombus luhuanus pedal retractor muscle Electrophoretic Forms ofStrombus luhuanus by different enzymes. These Octopine Dehydrogenase are catalyzed enzymes can be distinguished on the basis The distribution of the three electropho of ammonium sulfate precipitation, electro retic forms ofoctopine dehydrogenase among phoretic migration, ion exchange chromatog- Dehydrogenases in Strombidae-BALDwlN AND ENGLAND 391 raphy, and gel filtration. In addition, antisera and Opie 1978). This suggests that pyruvate produced against S. luhuanus octopine dehy inhibition per se is of limited value for pre drogenase (Baldwin, this issue) inhibit octo dicting the direction ofthe reactions catalyzed pine dehydrogenase but have no effect on the by the Strombus luhuanus enzymes in vivo. strombinejalanopine dehydrogenase activity. Both enzymes show similar K m values for Strombine dehydrogenase and alanopine pyruvate and NADH when determined at dehydrogenase activities copurify through all physiological concentrations of the cosub stages ofthe isolation procedure, maintaining strates (Tables 3, 5, 8). The pyruvate concen a constant activity ratio of 1 : 0.8 when 200 trations measured in pedal retractor muscles mM glycine and 200 mM alanine are used of resting animals are well below the Km as substrates. The identical electrophoretic values, while the NADH concentration is un migration rates obtained when D-strombine known. The K m values for glycine and arginine orMS-alanopine are used as substrates for the approximate the physiological levels of these reverse reaction provide further evidence that substrates, but the concentration ofalanine is both strombine and alanopine dehydrogenase one-tenth the Km value. Thus, glycine, rather activities are catalyzed by a single enzyme. than alanine, may serve as the major substrate for the alanopine dehydrogenase reaction, with strombine as the major product. Ifthis is Kinetic Properties ofAlanopine the case, then the activities of both octopine Dehydrogenase and Octopine Dehydrogenase dehydrogenase and alanopine dehydrogenase The kinetic properties of the two enzymes may be regulated by the rate at which pyru were investigated to provide information on vate and NADH are produced, thereby link the probable directions of the reactions in ing both reactions directly to the glycolytic vivo and on possible competition for common flux during muscle anoxia. In addition, simul substrates. taneous increases in pyruvate from glycolysis The pH profiles obtained for both enzymes and arginine from the hydrolysis of arginine suggest that the forward reactions are favored phosphate may accelerate octopine pro throughout the physiological pH range. duction by increasing the affinity of octopine Neither enzyme displayed substrate inhibi dehydrogenase for both substrates. tion for the forward reaction. With lactate For the reverse reaction, the low Km value dehydrogenases, pyruvate inhibition has been for octopine relative to octopine concentra used as a criterion for determining the direc tions in the exercised muscle may indicate that tion of the reaction in vivo (Dawson, Good octopine dehydrogenase is better suited for fellow, and Kaplan 1964; Long, Ellington, and the removal of the product of the forward Duda 1979). Storey and Storey (1979a) dis reaction following anaerobic muscle work tinguished between the physiological roles of than is alanopine dehydrogenase. mantle muscle and brain octopine dehydro genase in the cephalopod Sepia officinalis on Production ofStrombinejAlanopine and the basis of inhibition ofthe forward reaction Octopine in the Pedal Retractor Muscles of at high pyruvate concentrations. The pyru Strombidae vate saturation curves for Strombus luhuanus octopine dehydrogenase and alanopine dehy During bursts of rapid leaping, both drogenase resemble the Sepia mantle enzyme, strombinejalanopine and octopine accumu which is known to function in the direction of late together in the pedal retractor muscles of octopine formation during swimming (Storey Strombus luhuanus, S. gibberulus, and Lambis and ~torey 1979b). However, octopine dehy lambis, while arginine phosphate concentra drogenase from the fast adductor muscle of tions fall (Table 9). These results show that the scallop Pecten alba, which also produces the rate at which ATP is used during exercise octopine during rapid contractions ofthe fast exceeds the aerobic capabilities ofthe muscle. adductor muscle, is strongly inhibited at pyru Under these conditions, additional ATP is vate concentrations above I mM (Baldwin obtained from the breakdown of arginine 392 PACIFIC SCIENCE, Volume 36, July 1982

phosphate and from anaerobic glycolysis, tive inhibition ofoctopine dehydrogenase fol with both alanopine dehydrogenase and lowing exercise. One way of achieving this octopine dehydrogenase apparently acting to would be through inhibition ofoctopine dehy catalyze the terminal step in the glycolytic drogenase, but not alanopine dehydrogenase pathway. by-products of the two pyruvate reductase Some mollusk muscles with limited aerobic reactions, once these had accumulated to capabilities use anaerobic glycolysis in regen sufficiently high concentrations during muscle

erating arginine phosphate and reestablishing work. The K j values listed for these products energy charge during recovery from exercise (Tables 4, 6) show that octopine is a potent (Baldwin et al. 1981; Gade, Weeda, and inhibitor ofthe forward reaction catalyzed by Gabbott 1978; Grieshaber 1978). In the pedal octopine dehydrogenase, but has little effect retractor muscle of Nassarius coronatus, on alanopine dehydrogenase. For octopine which has high activities of both octopine dehydrogenase, the inhibition constant for dehydrogenase and lactate dehydrogenase octopine is about one-twelfth the concentra (Table I), only octopine is produced during tion ofoctopine measured following exercise. activity and only lactate during the recovery This inhibition is competitive with respect to phase (Baldwin et al. 1981). To determine the both substrates, with physiological concen relative contributions of alanopine dehydro trations of octopine (2 mM) increasing Km genase and octopine dehydrogenase during arginine about twelvefold and Km pyruvate recovery from exercise, metabolite levels were seventeenfold (Table 7). measured in pedal retractor muscles of The inhibitor constant for NAD+ is also Strombus luhuanus at rest, immediately fol lower for octopine dehydrogenase than for lowing exercise, and after various periods of alanopine dehydrogenase. If NAD+ concen recovery in water and in air (Table 10). trations in the pedal retractor muscle are In animals left to recover in aerated similar to other tissues (about I mM; Edington seawater following exercise, octopine and 1970, Tischler et al. 1977), the low K i NAD+ arginine phosphate concentrations remained values indicate that the cytoplasmic NAD+ / at postexercise levels, energy charge fell NADH ratio may also play a major role in slightly, and the concentration of strombine/ regulating the activities ofthese two dehydro alanopine increased with time. These results genases. indicate that anaerobic glycolysis is used Finally, while product inhibition provides during the recovery phase, but with only a possible explanation for the accumulation alanopine dehydrogenase catalyzing the ter of both octopine and strombine/alanopine minal reaction. A similar conclusion can be during exercise but only strombine/alanopine drawn from the results obtained for animals during recovery from exercise, it is not at left to recover in air. When exposed to air, all clear why two different enzymes are used the gills cannot be irrigated with water, and to catalyze the terminal step of anaerobic oxygen uptake may be impaired. In this situa glycolysis in the pedal retractor muscle of tion, the rate of anaerobic glycolysis ap Strombidae. Similar problems exist concern parently was not sufficient to maintain energy ing the octopine dehydrogenases and lactate charge and arginine phosphate concentra dehydrogenases that function independently tion at postexercise levels. Both arginine during different physiological states asso phosphate and energy charge fell, octopine ciated with anoxia in the pedal retractor levels remained constant, while strombine/ muscle of the gastropod Nassarius coronatus alanopine concentrations increased to the (Baldwin et al. 1981) and the foot of the bi highest values observed in this study. valve Cardium tuberculatum (Gade 1980). An important question arising from these results relates to the independent regulation of ACKNOWLEDGMENTS octopine dehydrogenase and alanopine dehy drogenase activity during the recovery period. We thank members of the Alpha Helix Presumably, mechanisms exist for the selec- Philippines expedition for providing many

, , ,~_ •• - .,~ • 'L "~ ." It ,,,. "-" , _., "'," ,.-,,, , "',,' • r _, , '~"~"""'_ Dehydrogenases in Strombidae-BALDwIN AND ENGLAND 393

of the mollusks used in this study, and the muscle metabolism. Can. J. Zooi. 54: 871 Heron Island Research Station for making 878. their facilities available to us. Strombine and FIELDS, J. H. A., H. GUDERLEY, K. B. STOREY, alanopine were kindly provided by J. Tempe. and P. W. HOCHACHKA. 1976b. The pyru vate branch point in squid brain: Compe LITERATURE CITED tition between octopine dehydrogenase and lactate dehydrogenase. Can. J. Zooi. ALP, P. R., E. A. NEWSHOLME, and V. A. 54: 879-885. ZAMMIT. 1976. Activities of citrate syn FIELDS, J. H. A., A. K. ENG, W. D. RAMSDEN, thase and NAD+ -linked and NADP-linked P. W. HOCHACHKA, and B. WEINSTEIN. isocitrate dehydrogenase in muscles from 1980. Alanopine and strombine are novel vertebrates and invertebrates. Biochem. J. iminoacids produced by a dehydrogenase 154: 689-700. found in the adductor muscle ofthe oyster, ATKINSON, D. E. 1977. Cellular energy meta Crassostreagigas. Arch. Biochem. Biophys. bolism and its regulation. Academic Press, 201: 110-114. New York. GADE, G. 1980. The energy metabolism of BALDWIN, J., and A. M. OPIE. 1978. On the the foot muscle of the jumping cockle, role of octopine dehydrogenase in the ad Cardium ruberculatum: Sustained anoxia ductor muscles of bivalve molluscs. Compo versus muscular activity. J. Compo Physioi. Biochem. Physiol. 61 B:85-92. 137B: 177-182. BALDWIN, J., A. K. LEE, and W. R. ENGLAND. GADE, G., and M. GRIESHABER. 1975. A rapid 1981. The functions of octopine dehydro and specific enzymatic method for the esti genase and D-Iactate dehydrogenase in the mation of L-arginine. Anal. Biochem. 66: pedal retractor muscle of the dog whelk 393-399. Nassarius coronatus (Gastropoda: Nas GADE, G., E. WEEDA, and P. A. GABBOTT. sariidae). Mar. BioI. 62: 235-238. 1978. Changes in the level of octopine BEAUMONT, A. R., T. R. DAY, and G. GADE. during the escape response of the scallop, 1980. Genetic variation at the octopine de Pecten maximus (L.). J. Compo Physiol. hydrogenase locus in the adductor muscle 124B: 121-127. of Cerastoderma edule (L.) and six other GRIESHABER, M. 1978. Breakdown and for bivalve species. Mar. BioI. Lett. 1: 137-148. mation of high-energy phosphates and DAWSON, D. M., T. L. GOODFELLOW, and octopine in the adductor muscle ofthe scal N. O. KAPLAN. 1964. Lactic dehydrogenase: lop, Chlamys opercularis (L.), during escape Functions of the two types. Science 143: swimming and recovery. J. Compo Physioi. 929-933. 126B:269-276. EDINGTON, D. W. 1970. Pyridine nucleotide GRIESHABER, M., and G. GADE. 1976. The oxidized to reduced ratio as a regulator of biological role of octopine in the squid, muscular performance. Experientia 26 : Loligo vulgaris (Lamarck). J. Compo 601-602. Physioi. 108B: 225-232. FIELDS, J. H. A. 1976. A dehydrogenase re HOCHACHKA, P. W., P. H. HARTLINE, and quiring alanine and pyruvate as substrates J. H. A. FIELDS. 1977. Octopine as an end from oyster adductor muscle. Fed. Proc. product of anaerobic glycolysis in the 35: 1687. chambered nautilus. Science 195: 72-74. FIELDS, J. H. A., and P. W. HOCHACHKA. 1981. HOCHACHKA, P. W., T. W. MOON, Purification and properties of alanopine T. MUSTAFA, and K. B. STOREY. 1975. dehydrogenase from the adductor muscle Metabolic sources of power for mantle of the oyster, Crassostrea gigas (Mollusca, muscle of a fast swimming squid. Compo Bivalvia). Eur. J. Biochem. 114:615-621. Biochem. Physioi. 52B: 151-157. FIELDS, J. H. A., J. BALDWIN, and P. W. LAYNE, E. 1957. Spectrophotometric and tur HOCHACHKA. 1976a. On the role of octo bidometric methods for measuring pro pine dehydrogenase in cephalopod mantle teins. Pages 447-454 in S. P. Colowick and

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N. O. Kaplan, eds. Methods in enzymo isozymes of lactate dehydrogenase. Eur. logy. Vol. 3. Academic Press, New York. J. Biochem. 93: 545-552. LONG, G. L., W. R. ELLINGTON, and T. F. ---. 1979b. Octopine metabolism in the DUDA. 1979. Comparative enzymology and cuttle fish, Sepia offieinalis: Octopine pro physiological role of D-lactate dehy duction by muscle and its role as an aerobic drogenase from the foot muscle of two substrate for non-muscular tissues. gastropod molluscs. J. Exp. Zool. 207: J. Compo Physiol. l31B:311-319. 237-248. TISCHLER, M. E., D. FRIEDRICHS, K. COLL, OLOMUCKI, A, C. Huc, F. LEFEBURE, and and J. R. WILLIAMSON. 1977. Pyridine nuc N. VAN THOAI. 1972. Octopine dehydro leotide distributions and enzyme mass genase. Evidence for a single-chain struc action ratios in hepatocytes from fed and ture. Eur. J. Biochem. 28:261-268. starved rats. Arch. Biochem. Biophys. REGNOUF, F., and N. VAN THOAI. 1970. 184:222-236. Octopine and lactate dehydrogenases in ZAMMIT, V. A, and E. A NEWSHOLME. 1976. mollusc muscles. Compo Biochem. Physiol. The maximum activities of hexokinase, 32:411-416. phosphorylase, phosphofructokinase, glyc SANGSTER, A W., S. E. THOMAS, and N. L. erol phosphate dehydrogenases, lactate TINGLING. 1975. Fish attractants from dehydrogenase, octopine dehydrogenase, marine invertebrates. Arcamine from Area phosphoenolpyruvate carboxykinase, nuc zebra and strombine from Strombus gigas. leoside diphosphatekinase, glutamate oxalo Tetrahedron 31: 1135-1137. acetate transaminase and argine kinase STOREY, K. B. 1977. Tissue specific isozymes in relation to carbohydrate utilization ofoctopine dehydrogenase in the cuttle fish, in muscles from marine invertebrates. Sepia officinalis. The roles of octopine de Biochem. J. 160:447-462. hydrogenase and lactate dehydrogenase in DE ZWAAN, A, R. J. THOMPSON, and D. R. Sepia. J. Compo Physiol. 115B: 159-169. LIVINGSTONE. 1980. Physiological and bio STOREY, K. B., and J. M. STOREY. 1979a. chemical aspects ofthe valve snap and valve Kinetic characterization of tissue-specific closure responses in the giant scallop isozymes of octopine dehydrogenase from Plaeopeeten magellanieus. II. Biochemistry. mantle muscle and brain ofSepia offieinalis. J. Compo Physiol. 137B: 105-114.

Functional similarities to the M 4 and H4