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

An Immunochemical Study of Structural and Evolutionary Relationships among Molluscan Octopine Dehydrogenases1

JOHN BALDWIN2

ABSTRACT: Antisera produced against octopine dehydrogenases isolated from a gastropod and a cephalopod were used to investigate structural and evolutionary relationships of this enzyme in a range of mollusks. Antisera against octopine dehydrogenase of the blue-ringed octopus Hapalochlaena maculosa was most effective in inhibiting the enzyme from other octopods, followed by the enzymes of squids and cuttlefishes. Limited inhibition also occurred with octopine dehydrogenase ofNautilus pompilius, a representative of the most ancient group of living cephalopods. This antisera did not inhibit octopine dehydrogenases ofgastropods or bivalves. Antisera against the enzyme of the gastropod Strombus luhuanus inhibited octopine dehydrogenases from other genera ofthe family Strombidae, but did not inhibit the enzyme from other families of gastropods or the enzymes from cephalopods or bivalves. It is concluded that the octopine dehydrogenases of cephalopods possess structural similarities and have diverged from a common ancestral gene. The structural and evolutionary relationships among gastropod octopine dehydrogenases and the relationships among octopine dehydrogenases from different molluscan classes remain unresolved.

OCTOPINE DEHYDROGENASE (EC 1.5.1.11) genase in regenerating cytoplasmic NAD+ occurs in many mollusks and several other during temporary anoxia associated with invertebrate phyla, and also in the crown gall bursts ofrapid locomotion (Baldwin, Lee, and tumors ofplants where it is incorporated by a England 1981; Giide 1980). Octopine in plant bacterial plasmid (Baldwin and England, this crown gall tissue may serve as a carbon or issue; Ellington 1979; Giide 1980; Goldmann nitrogen source for oncogenic agrobacteria 1977; Hass et al. 1973; Regnoufand van Thoai (Drummond 1979). 1970; Zammit and Newsholme 1976). The The various invertebrate and plant octo­ enzyme is unusual among dehydrogenases in pine dehydrogenases possess structural and being monomeric, with a molecular weight kinetic similarities; therefore, it is ofinterest to resembling the subunit size of most multi­ speculate on possible evolutionary relation­ meric dehydrogenases (Baldwin and England, ships among these enzymes. In this study, an this issue; Fields, Baldwin, and Hochachka immunochemical approach has been taken to 1976a; Goldmann 1977; Olomucki et al. investigate structural and evolutionary re­ 1972). lationships among the octopine dehydro­ In mollusk muscle, the octopine dehydro­ genases of cephalopod, gastropod, and genase reaction can replace lactate dehydro- bivalve mollusks.

1 This research was conducted as part of the Alpha Helix Cephalopod Expedition to the Republic of the MATERIALS AND METHODS Philippines, supported by National Science Foundation grant PCM 77-16269 to J. Arnold. This study was also supported in part by the Australian Research Grants Experimental Animals Committee. 2 Monash University, Department of Zoology, Blue-ringed octopuses, Hapalochlaena Clayton, Victoria 3168, Australia. maculosa, were captured by dredging on 357 358 PACIFIC SCIENCE, Volume 36, July 1982 mussel beds in Port Phillip Bay, Victoria, Aus­ est specific activity were concentrated by tralia. Strombus luhuanus were collected on membrane filtration (Amicon Diaflo, PMIO) the reef flat at Heron Island, Queensland, and further purified by gel filtration on Australia. Muscles from these animals were Sephadex G 100 equilibrated with 50 mM frozen in liquid nitrogen immediately after Tris-HCl buffer, pH 7.6, containing 0.1 mM death and stored at - 20°C for up to 6 months EDTA, 0.1 mM DTE, and 10% glycerol. The without loss of octopine dehydrogenase ac­ most active fractions were pooled, concen­ tivity. This provided the material from which trated by membrane filtration, and stored at cephalopod and gastropod octopine dehydro­ 4°C in 80% saturated ammonium sulfate. genases were isolated for antibody produc­ Octopine dehydrogenase was purified from tion. Pecten alba came from Port Phillip Bay, the pedal retractor muscle of Strombus while other mollusks used in the study were luhuanus as described by Baldwin and collected in the Republic of the Philippines England (this issue). during the Alpha Helix expedition. Production ofAntisera Isolation ofDctopine Dehydrogenases for Antisera against octopine dehydrogenases Antibody Production isolated from the blue-ringed octopus and Frozen mantle and arm muscle ofthe blue­ Strombus luhuanus were produced in adult ringed octopus was homogenized in 10 vol New Zealand white rabbits. On day 0, 3 mg of of ice-cold buffer [25 mM Tris-HCl, 0.1 mM protein in 1 ml of 20 mM sodium phos­ ethylenediaminetetraacetic acid (EDTA), 0.1 phate/50 mM sodium chloride buffer, pH 7.4, mM 1,4-dithioerythritol (DTE), 10% glyc­ was emulsified with an equal volume of erol, pH 8.5] using a Sorvall omni-mixer. The Freund's complete adjuvant3 and injected homogenate was centrifuged at 20,000 x g subcutaneously at multiple sites in the hind for 30 min at 4°C, and the pellet discarded. leg. This procedure was repeated twice at The portion of the supernatant precipitating intervals of7 days. A final injection of 1 mg of between 35 and 75% ammonium sulfate satu­ protein in 1.5 ml of buffer was administered ration was collected by centrifugation, dis­ on day 21. Blood was collected from the mar­ solved in a small volume ofthe homogenizing ginal ear vein each week from day 28. Serum buffer, and dialyzed against a large volume of from the clotted blood was centrifuged and this buffer. The dialyzed sample was applied stored at - 20°C in 0.5-ml aliquots. A sample to a D-(diethylaminoethyl) (DEAE) cellulose of control serum was collected from each column (Whatman DE22) equilibrated with rabbit prior to the initial injection ofoctopine the homogenizing buffer, and octopine de­ dehydrogenase. hydrogenase activity was washed through with this buffer. Pooled fractions from the Preparation ofDctopine Dehydrogenase DEAE column were concentrated by ammo­ Extracts for Immunochemical Titration nium sulfate precipitation and dialyzed against 10 mM sodium phosphate buffer, pH Mantle muscle from octopods, squids, and 6.6, containing 0.1 mM EDTA, 0.1 mM DTE, cuttlefishes; spadix of Nautilus; pedal retrac­ and 10% glycerol. The dialyzed sample was tor muscle of gastropods; and adductor mus­ loaded onto a D-(carboxymethyl) (CM) cel­ cles of bivalves were homogenized in 10 vol lulose column (Whatman CM23) equilibrated of ice-cold phosphate buffer (50 mM sodium with the dialysis buffer. Octopine dehydro­ phosphate, 0.1 mM EDTA, 0.1 mM DTE, genase activity was eluted by washing with the 10% glycerol, pH 7.0) and centrifuged at equilibration buffer. Fractions with the high- 20,000 x g for 30 min at 4°C. The super­ natants were stored at - 20°C and used as

3 Mineral oil 85% (v/v), mannide monooleate 15% a source of octopine dehydrogenase for im­ (v/v), M. tuberculosis (heat-killed) 0.5 mg/ml. munochemical titrations. Evolution of Molluscan Dehydrogenases-BALDWIN 359

Electrophoresis centage inhibition of the enzyme by the antiserum. Octopine dehydrogenase extracts were ex­ amined by electrophoresis on cellulose acetate gels (Cellogel, Chemtron, Milano) using 75 mM Tris-citrate buffer, pH 7.5. The gels were RESULTS AND DISCUSSION stained for octopine dehydrogenase activity as The inhibition curves (Figures 1, 2) show described by Fields et a!. (1976b). the effects ofincreasing amounts ofantiserum on the activity of octopine dehydrogenases Assay ofOctopine Dehydrogenase Activity from various mollusks. The relationship be­ tween the percentage inhibition and the Octopine dehydrogenase activity was de­ volume of antiserum added is linear only in termined at 340 nm with a Zeiss DM 4 record­ the region of the curve where enzyme in­ ing spectrophotometer. The cell temperature hibition is less than about 40%. To obtain was maintained at 25°C with a circulating numerical inhibition values for comparing the water bath. Assays were carried out with from different enzymes tested, the initial linear por­ 5 to 25 J.LI ofsample in a total volume of 1 m!. tion of each curve was extrapolated to the The reaction mixture, which was selected to bottom axis. This inhibition value corre­ give maximum activity with most ofthe tissue sponds to the number of microliters of anti­ extracts examined, contained 5 mM sodium serum that would have inhibited one unit of pyruvate, 20 mM arginine, and 0.2 mM octopine dehydrogenase activity if the re­ NADH in 50 mM sodium phosphate buffer, lationship between percentage inhibition and pH 7.0. The rate measured with this assay volume of antiserum had remained linear mixture is equal to the sum of the activities (Jokay and Toth 1966, Mochizuki and Hori ofboth octopine and lactate dehydrogenases, 1980). and lactate dehydrogenase was potentially Electrophoresis gave a single major ano­ present in both the mollusk muscle extracts dally migrating band of octopine dehydro­ and the rabbit serum. Octopine dehydro­ genase activity for each ofthe cephalopod and genase values were corrected by subtracting gastropod muscle extracts in which enzyme the rate due to lactate dehydrogenase alone activity was inhibited by antisera. It is as­ when arginine was omitted from the assay. sumed that the inhibition curves reflect inter­ One unit of enzyme activity was defined as actions between the antiserum and a single the amount required to oxidize 1 J.Lmole molecular form of octopine dehydrogenase. NADH/min. Results obtained with antiserum prepared against octopine dehydrogenase of the blue­ Immunochemical Titration ofOctopine ringed octopus are shown in Figure I and Dehydrogenase in Mollusk Muscle Extracts Table 1. This antiserum was most effective in inhibiting the enzymes from other octopods, Appropriate volumes of muscle extract followed by the enzymes of squids and cuttle­ containing 0.5 IV of octopine dehydrogenase fishes. More limited inhibition occurred with activity were mixed with varying amounts of octopine dehydrogenase from the nautiloid, antiserum, control serum, and buffer (50 mM Nautilus pompilius, a representative of the sodium phosphate, 0.1 mM EDTA, 0.1 mM most ancient group ofliving cephalopods. The DTE, 10% glycerol, pH 7.0) such that the relative inhibition potencies reflect the gener­ serum concentration was kept constant in a ally accepted phylogenetic relationships final volume of 100 J.L!' The samples were in­ among the subclasses and orders of cepha­ cubated for 1hr at 25°C, 16 hr at 4°C, and then lopods examined (Donovan 1977). This anti­ centrifuged at 20,000 x g for 30 min at 4°C. serum did not inhibit octopine dehydrogenases The supernatants were assayed for octopine from the five species of gastropods and two dehydrogenase activity to determine the per- species of bivalves tested.

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TABLE I 100 IMMUNOCHEMICAL TITRATION OF OCTOPINE DEHYDROGENASE ACTIVITY IN MOLLUSK MUSCLE EXTRACTS WITH ANTISERUM PRODUCED AGAINST OCTOPINE DEHYDROGENASE OF THE BLUE-RINGED 80 \ OCTOPUS, Hapalochlaena maculosa RELATIVE ;!. \\\ . INHIBITION INHIBITION 60 SPECIES VALUE* POTENCyt £ .\ \ > \ . Octopods u 0 Hapalochlaena maculosa 65 I O. membranaceus 80 0.813 .~ 40 '" \, 160 0.406 '0 O. macropus a; \\ 180 0.361 O. horridus a:: ~ Squids \. 0.245 Thysanoteuthis rhombus 265 20 \\ • Symplectoteuthis • oualaniensis 300 0.217 \. Sepioteuthis lessoniana 310 0.210 Cuttlefishes Sepia latimanus 320 0.203 200 400 600 800 o S. bandensis 390 0.167 ).II ant'tserum / un·,t enzyme act'tvity Nautiloid Nautilus pompilius 1,200 0.054 FIGURE I. Inhibition curves of cephalopod octopine dehydrogenases with antiserum produced against Gastropods N.\. octopine dehydrogenase of the blue-ringed octopus. Strombus sinuatus NJ. LEGEND: ", Octopus macropus; ., Thysanoteuthis rhom­ Lambis scorpius N.\. bus; +, Sepia bandensis; ., Nautilus pompilius. Tibia Nassarius coronatus N.\. Cerithium nodulosium NJ. Bivalves Pecten alba N.\. 100 Periglypta reticulata N.\.

*Values in microliters of serum required to inhibit 1 unit of enzyme .activity. N.I. indicates no inhibition. 80 tInhibition value relative to Hapalochlaena mocu/osa octopine dehydro­ genase. - ~ $. Antiserum produced against octopine de­ 60 "- J~ hydrogenase of the gastropod Strombus :~ u luhuanus (Figure 2, Table 2) inhibited the 0 enzyme from the three genera of the family ~ 40 ~ 0 -.; Strombidae tested. The results did not clearly 0: ~ \ distinguish between octopine dehydrogenases 20 of Strombus and Lambis. However, the rel­ i\ .~. ative inhibition potency value obtained for \\ octopine dehydrogenase of Tibia shows that I. this enzyme is antigenically quite distinct 50 100 150 200 250 300 350 400 J.I1 anflserum/ unit enzyme OCt"IV"lty from the enzymes of the other two genera. This antiserum had no inhibitory effect on the FIGURE 2. Inhibition curves of gastropod octopine octopine dehydrogenases from the other five dehydrogenases with antiserum produced against octo­ families of gastropods tested, or on the pine dehydrogenase of Strombus luhuanus. LEGEND: ", Strombus canarium; ., Lambis chiragra; ., Tibia enzymes from cephalopods or bivalves. martinii; ., Cerithium nodulosium. Estimating structural and evolutionary re- Evolution of Molluscan Dehydrogenases-BALDWIN 361

TABLE 2

IMMUNOCHEMICAL TITRATION OF OCTOPINE DEHYDROGENASE ACTIVITY IN MOLLUSK MUSCLE EXTRACTS WITH ANTISERUM PRODUCED AGAINST OCTOPINE DEHYDROGENASE OF Strombus luhuanus

RELATIVE INHIBITION INHIBITION SPECIES VALUE* POTENCyt

Gastropods Family Strombidae, Strombus luhuanus 20 1 S. canarium 21 0.952 S.labiatus 21 0.952 S. aurisdianae 24 0.833 S. sinuatus 25 0.800 S. lentiginosus 27 0.741 Lambis scorpius 22 0.909 L. lambis 22 0.909 L. chiragra 26 0.769 L. mi//epeda 28 0.714 Tibia martinii 300 0.067 Family Buccinidae, Cantharus undosus N.!. Family Bursidae, Bursa sp. N.J. Family Muricidae, Murex tribulus N.!. Family Nassariidae, Nassarius coronatus N.J. Family Cerithiidae, Cerithium nodulosium N.!. Cephalopods Hapaloch/aena maculosa N.J. Sepioteuthis lessoniana N.J. Sepia latimanus N.J. Nautilus pompi/ius N.!. Bivalves Pecten alba N.!. Periglypta reticulata N.!.

• Values in microliters of serum required to inhibit 1 unit ofenzyme activity. N.J. indicates no inhibition. t Inhibition value relative to Strombus luhuanus octopine dehydrogenase.

lationships among enzymes by immuno­ (amount of substrate transformed per unit chemical inhibition has proved successful with time per enzyme molecule). For example, in a range ofenzymes from both vertebrates and a group of homologous enzymes containing invertebrates (Matsuoka and Hori 1980, identical antigenic sites but differing in turn­ Mochizuki and Hori 1980, Sado and Hori over number, enzymes with highest turnover 1978). A major advantage of this method numbers will appear to be most susceptible to when comparing enzymes from a large inhibition. This is because the ratio between number of organisms is that it requires only the volume of antiserum added and the isolation ofthe enzymes used to produce anti­ number of enzyme molecules in the assay sera. Even if these enzymes are not purified to system is greater per unit of enzyme activity. homogeneity, the specificity of the enzyme These potential problems associated with dif­ assay system should avoid problems as­ ferences in turnover number do not appear to sociated with interactions of the antiserum be significant in the present study. The order­ with other proteins in the tissue homogenates ing of relative inhibition potencies obtained tested. However, difficulties may arise with for both the cephalopod and gastropod oc­ this technique because differences in relative topine dehydrogenases follow the accepted inhibition potency among homologous en­ phylogenetic relationships among the animals zymes may reflect not only structural differ­ tested. This suggests that the accumulation of ences, but also differences in turnover number structural changes in the enzyme with time 362 PACIFIC SCIENCE, Volume 36, July 1982

has not been masked by differences in turn­ ACKNOWLEDGMENTS over number. The author thanks members of the Alpha The results obtained in this study show that Helix Philippines expedition for providing the octopine dehydrogenases of octopods, and identifying many of the mollusks used, squids, cuttlefishes, and nautiloids are struc­ and the Heron Island Research Station for turally similar in that they share common providing facilities during this study. antigenic determinants. Presumably, these en­ zymes are homologous, having diverged from a common ancestral gene that was present in LITERATURE CITED the class Cephalopoda prior to the separation of the subclasses Nautiloidea and Coleoidea. BALDWIN, J., A. K. LEE, and W. R. ENGLAND. Evidence from the fossil record indicates that 1981. The functions of octopine dehydro­ this separation had already occurred by the genase and D-lactate dehydrogenase in the early Carboniferous (Donovan 1977, Stasek pedal retractor muscle of the dog whelk 1972). Nassarius coronatus (Gastropoda: Nas­ More limited information was obtained on sariidae). Mar. Bio!. 62: 235-238. the relationships among gastropod octopine DONOVAN, D. T. 1977. Evolution of the di­ dehydrogenases. The enzymes from the three branchiate cephalopoda. Symp. Zoo!. Soc. genera of the family Strombidae clearly Lond. 38: 15-48. possess structural similarities. However, little DRUMMOND, M. 1979. Crown gall disease. can be said of the structural relationships Nature 281: 343-346. among the octopine dehydrogenases of the ELLINGTON, W. R. 1979. Octopine dehydro­ Strombidae and the octopine dehydrogenases genase in the basilar muscle of the sea from other gastropod families, as the latter anemone Metridium senile. Compo were not inhibited by antisera produced Biochem. Physio!. 63B: 349-354. against the Strombus luhuanus enzyme. FIELDS, J. H. A, J. BALDWIN, and P. W. Similarly, failure of the antisera produced HOCHACHKA. 1976a. On the role ofoctopine against cephalopod or gastropod octopine de­ dehydrogenase in cephalopod mantle hydrogenases to inhibit the enzyme from muscle metabolism. Can. J. Zoo!. 54:871­ other molluscan classes leaves unresolved the 878. structural and evolutionary relationships FIELDS, J. H. A, H. GUDERLEY, K. B. STOREY, among octopine dehydrogenases of cepha­ and P. W. HOCHACHKA. 1976b. The py­ lopods, gastropods, and bivalves. The extant ruvate branch point in squid brain: Com­ molluscan classes are considered to have di­ petition between octopine dehydrogenase verged from some common ancestral group and lactate dehydrogenase. Can. J. Zoo!' during the Precambrian, but evidence ofthese 54: 879-885. events is lacking from the fossil record. Stasek GADE, G. 1980. Biological role of octopine (1972 :3) has proposed that the stem groups formation in marine molluscs. Mar. Bio!. that gave rise to the antecedents of mollusks, Lett. 1: 121-135. annelids, and arthropods "could have been GOLDMANN, A. 1977. Octopine and nopaline placed in an expanded concept of the turbel­ dehydrogenases in crown-gall tumors. larian Platyhelminthes or in a single, now de­ Plant Sci. Lett. 10: 49-58. funct, phylum that would have included the HASS, S., F. THOMBE-BEAU, A OLOMUCKI, and extant Nemertinea." It is ofinterest when con­ N. VAN THOAI. 1973. Purification de sidering the evolutionary relationships of l'octopine deshydrogenase de Sipunculus molluscan octopine dehydrogenases that oc­ nudus. Compt. Rend. Hebd. Seane. Acad. topine has been found in the nemerteans Sci. (Paris) 276: 831-834. Cerebratulus occidentalis and Lineus picti­ JOKAY, I., and S. TOTH. 1966. Microquantita­ frons (Robin 1964), and that octopine dehy­ tive antibody determination by means of drogenase occurs in the sipunculid Sipunculus phosphorylase antigen. 1. Determination of nudus (van Thoai and Robin 1959). circulating antibodies in roosters. Zeit. Evolution of Molluscan Dehydrogenases-BALDWIN 363

Immunforsch. expo Ther. 130: 17-39. logical relatedness of glucose-6-phosphate MATSUOKA, N., and S. H. HORi. 1980. dehydrogenases from vertebrate and inver­ Immunological relatedness of hexose-6­ tebrate species. Japan. J. Genetics 53: 91­ phosphate dehydrogenase and glucose-6­ 102. phosphate dehydrogenase in echinoderms. STASEK, C. R. 1972. The molluscan frame­ Compo Biochem. Physio!. 65B: 191-198. work. Pages 1-44 in M. Florkin and B. J. MOCHIZUKI, Y, and S. H. HORi. 1980. Scheer, eds. Chemical zoology. Vo!. 7. Immunological relationships of starfish Academic Press, New York. hexokinases: Phylogenetic implication. VAN THOAI, N., and Y ROBIN. 1959. Meta­ Compo Biochem. Physio!. 65B: 119-125. bolisme des derives guanidyles. VIII. Bio­ OLOMUCKI, A., C. Huc, F. LEFEBURE, and synthese de I'octopine et repartition de N. VAN THOAi. 1972. Octopine dehydro­ I'enzyme I'operant chez les invertebres. genase. Evidence for a single-chain struc­ Biochim. Biophys. Acta 35: 446-453. ture. Eur. J. Biochem. 28: 261-268. ZAMMIT, V. A., and E. A. NEWSHOLME. 1976. REGNOUF, F., and N. VAN THOAi. 1970. The maximum activities of hexokinase, Octopine and lactate dehydrogenases in phosphorylase, phosphofructokinase, glyc­ mollusc muscles. Compo Biochem. Physio!. erol phosphate dehydrogenases, lactate 32:411-416. dehydrogenase, octopine dehydrogenase, ROBIN, Y 1964. Biological distribution of phosphoenolpyruvate carboxykinase, nuc­ guanidines and phosphagens in marine an­ leoside diphosphatekinase, glutamate nelida and related phyla from California oxaloacetate transaminase and arginine with a note on pluriphosphagens. Compo kinase in relation to carbohydrate utiliza­ Biochem. Physio!. 12: 347-367. tion in muscles from marine invertebrates. SADO, Y., and S. H. HORi. 1978. Immuno- Biochem. J. 160:447-462.

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