Pacific Science (1982), vol. 36, no. 3 © 1983 by the University of Hawaii Press. All rights reserved 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