Journal J Comp Physiol (1982) 149 : 57-65 of Comparative Physiology- B Springer-Verlag 1982

Alanopine Dehydrogenase: Purification and Characterization of the from littorea Foot Muscle

William C. Plaxton and Kenneth B. Storey Institute of Biochemistry,Carleton University, Ottawa, Ontario, Canada K1S 5B6 Accepted June 12, 1982

Summary. 1. Alanopine dehydrogenase from the levels (pyruvate and alanine are products foot muscle of the common periwinkle, Littorina of glycolysis) and decreasing pH (occurring during littorea was purified to homogeneity using a com- anoxia) on enzyme apparent K,,'s for substrates bination of ammonium sulphate fractionation, gel would favour alanopine accumulation as a filtration and chromatofocusing. of anaerobic glycolysis. 2. The enzyme has a molecular weight of 42,200 + 500 and is a monomer. 3. L-Alanine and pyruvate are the preferred substrates. Alternate amino acids (>L-0c- aminobutyrate > L-serine > L-cysteine) are used at Introduction rates less than 37% of enzyme activity with L-ala- nine. Alternate keto acids used include oxaloace- Anaerobic energy production in vertebrate tissues tate, ~-ketobutyrate and glyoxylate. The enzyme depends upon the action of lactate dehydrogenase is specific for meso-alanopine in the reverse direc- to maintain cytoplasmic redox balance, lactic acid tion; D-strombine is not oxidized. accumulating as the product of carbohydrate fer- 4. Apparent K,,'s for both pyruvate and L-ala- mentation. The tissues of many marine inverte- nine decrease with increasing co-substrate (L-ala- brates, however, contain activities of one or more nine or pyruvate) concentration or with decreasing imino acid dehydrogenases: pH. pyruvate + amino acid + NADH + H + ~-imino 5. Absolute Kr,'s for pyruvate and L-alanine are acid + NAD + + H20, which functionally replace 0.17+0.02 and 14.9+0.85 mM at pH 6.5 rising to lactate dehydrogenase as the terminal enzyme of 0.26_+0.01 and 23.8+0.52 mM at pH 7.5, respec- anaerobic glycolysis (Regnouf and Thoai 1970; tively. Apparent Km's for meso-alanopine are Fields 1976; de Zwaan and Zurburg 1981 ; Dando 6.5 mM at pH 6.5 and 50 mM at pH 8.5 while ap- et al. 1981). Octopine dehydrogenase, catalyzing parent Km'S for NADH (9+0.1 gM) and NAD + the reductive condensation of pyruvate and L-argi- (0.18-t-0.03 raM) are pH independent. nine, has been well characterized (Thoai et al. 6. Substrate inhibition by pyruvate (Iso = 1969; Fields et al. 1976; Storey and Storey 1979 a); 8 raM) and L-alanine 05o=450-550 raM) occurs amongst molluscs, high speed swim- at saturating co-substrate levels while NAD + (Ki = ming, supported by glycogenolysis and arginine 0.16 +_ 0.012 raM) and meso-alanopine (Ki = 35 _+ phosphate breakdown, results in the accumulation 0.4 mM) are product inhibitors of the forward re- of octopine as the end product of mantle muscle action. ATP and ADP are competitive inhibitors metabolism (Grieshaber and G/ide 1976i S1Lorey with respect to NADH while L-lactate, D-strom- and Storey 1979b; Baldwin and England 1980). bine and succinate inhibit with respect to pyruvate Alanopine dehydrogenase, catalyzing the re- and L-alanine. action: 7. The kinetic properties of alanopine dehy- pyruvate + L-alanine + NADH + H + ,~- meso- drogenase favour enzyme function in cytoplasmic alanopine + NAD + + HzO, was first identified in redox balance during anoxia stress in this intertidal 1976 in the muscle tissues of the , gastropod. In particular the effects of rising co- gigas (Fields 1976) while alanopine (2,T-iminodi-

0174-1578/82/0149/0057/$01.80 58 W.C. Plaxton and K.B. Storey: Alanopine Dehydrogenase in Littorina Foot Muscle

propionic acid) and/or strombine (2-methylimino- Assays were started by the addition of enzyme preparation. diacetic acid) accumulate as end products of 14C- Standard assay conditions for lactate dehydrogenase were: glucose fermentation during anoxia in oyster ven- 50 mM imidazole buffer, pH 7.5, 1.3 mM pyruvate and 0.1 mM NADH. One unit of enzyme activity is defined as the amount tricle (Collicutt and Hochachka 1977). Subse- of enzyme utilizing 1 gmol NADH per minute at 23 ~ quently alanopine dehydrogenase and strombine Apparent Km values were determined from Hanes plots dehydrogenase (utilizing glycine as the amino acid under conditions of constant, saturating concentrations of co- substrate) activities have been identified in the tis- substrate(s) while absolute K m values for L-alanine and pyruvate sues of a variety of marine bivalve and gastropod were derived from secondary plots using data from apparent Km determinations at 3 subsaturating co-substrate concentra- molluscs as well as in sea anemones and other tions (Cornish-Bowden 1979). Inhibitor constants (Ki) were de- groups (de Zwaan and Zurburg 1981 ; Dando et al. termined from Dixon plots and the effect of inhibitors was 1981 ; Ellington 1979, 1981). Some kinetic proper- evaluated using double reciprocal plots. Iso's (inhibitor concen- ties of purified oyster adductor muscle alanopine tration producing 50% inhibition of enzyme activity) were de- termined by the method of Job et al. (1978). All kinetic parame- dehydrogenase have recently been reported (Fields ters are the means of duplicate determinations on three separate and Hochachka 1981); the enzyme has a broad preparations of the purified enzyme and are reproducible within amino acid specificity, utilizing L-alanine, glycine at least_+ 10%. In most cases, results are expressed as means_+ and several other amino acids at nearly equal rates s.e.m. and probably produces both alanopine and strom- bine in vivo. Purification of Alanopine Dehydrogenase. Foot muscle was minced with scissors and homogenized (1 : 5 w/v) for 2 x 1 min In the present study we have examined the ki- in ice-cold 50 mM imidazole buffer, pH 7.5 containing 15 mM netic properties of alanopine dehydrogenase puri- /%mercaptoethanol, using a Polytron PT 10-35 homogenizer. fied from foot muscle of the intertidal gastropod The homogenate was centrifuged at 27,000 x g for 20 min mollusc, Littorina littorea. The gastropod enzyme, at 4 ~ and the supernatant used as the source of crude enzyme. unlike bivalve, alanopine dehydrogenase, is highly The crude supernatant was brought to 2.95 M (60% satura- tion) with ammonium sulphate, stirred at room temperature specific for L-alanine and meso-alanopine as sub- for 30 rain, and then centrifuged as above. The protein pellet strates, a specificity which may link enzyme func- was discarded and the resulting supernatant was then adjusted tion in vivo to anaerobiosis, the production of to 4.03 M (80% saturation) with ammonium sulphate and again alanopine coupling two products, pyruvate and stirred and centrifuged. The pellet was resuspended in 2-3 ml 20 mM imidazole buffer, pH 7.4 containing 10 mM fl-mercap- L-alanine, of anaerobic glycolysis. Other kinetic toethanol and centrifuged to remove insoluble material. The characteristics of the enzyme including strong pH partially purified enzyme solution was then layered onto a col- effects on enzyme affinity for pyruvate and alanine umn (60 x 0.9 cm) of Sephadex G-100 equilibrated in 20 mM also appear designed to promote alanopine synthe- imidazole buffer, pH 7.4 containing 10 mM fl-mercaptoethanol. sis during anoxia. The column was eluted at 20 ml/h using the same buffer and 1 ml fractions were collected. Peak fractions, containing alanopine dehydrogenase activity were pooled and layered onto a column (30 x 0.9 cm) of PBE 94 chromatofocusing exchanger Materials and Methods pre-equilibrated in 20 mM imidazole buffer, pH 7.4 containing 10 mM/Lmercaptoethanol. The column was eluted at 20 ml/h and Chemicals. Specimens of the common periwinkle, with 200 ml Polybuffer 74 (stock diluted 1:8 with distilled Littorina littorea, were obtained from local markets. Foot water) adjusted to pH 4.0 and 1 ml fractions were collected. muscle was dissected out and stored at -80 ~ until use. No Alanopine dehydrogenase activity eluted in a single sharp peak loss of enzyme activity was seen during storage for periods at pH 5.7 (-I-0.05, n=3) on the linear pH gradient. Peak frac- of at least one month. tions from chromatofocusing were pooled and applied to a ATP, ADP, D- and Lqactate and NADH were purchased second, larger G-100 column (90x 1.5 cm) equilibrated in from Boehringer Mannheim Corp. while all other biochemicals 50 mM imidazole buffer, pH 7.5 containing 10 mM p-mercap- were from Sigma Chemical Co., Sephadex G-100, PBE 94 chro- toethanol and 0.04% sodium azide. The column was eluted matofocusing exchanger and Polybuffer 74 were obtained from with this same buffer and peak fractions, containing purified Pharmacia Fine Chemicals, Ampholines (pH 4-6) were from alanopine dehydrogenase, were combined and stored at 4 ~ LKB Products, and molecular weight standard proteins were for use in kinetic studies. The purified enzyme was stable in from Sigma and Pharmacia. this form, with no loss of activity or change in kinetic param- All buffers used in the study were adjusted to the respective eters, for at least 2 weeks. pH at 23 ~

Enzyme Assay and Kinetic Studies. Alanopine dehydrogenase Molecular Weight Determination. Molecular weight determina- activity was assayed by monitoring NAD(H) utilization at tions were made on a column (90 x 1.5 cm) of Sephadex G-100 340 nm using a Gilford recording spectrophotometer attached using 50raM imidazole buffer, pH 7.5 containing 10raM to a circulating water bath for temperature control of cuvettes. /~-mercaptoethanol and 0.04% sodium azide as the equilibra- Standard assay conditions for alanopine dehydrogenase were: tion/elution buffer. One ml fractions were collected and assayed 50 mM imidazole buffer, pH 7.5, 1.3 mM pyruvate, 130 mM for absorbance at 280 nm and alanopine dehydrogenase activ- L-alanine and 0.1 mM NADH in the forward direction, and ity. The molecular weight of alanopine dehydrogenase was 50 mM Tris-HC1 buffer, pH 9.2, 50 mM meso-alanopine and determined from a plot of K,~ (partition coefficient) versus log 2 mM NAD + in the reverse direction in a final volume of 1 ml. M r for the standard proteins: ribonuclease (Mr = 13,700), chy- W.C. Plaxton and K.B. Storey: Alanopine Dehydrogenase in Littorina Foot Muscle 59 motrypsinogen (M, = 25,000), ovalbumin (Mr=45,000) and Table 1. Summary of the purification of alanopine dehydroge- bovine serum albumin (Mr = 67,000). nase from the foot muscle of the gastropod mollusc, Littorina linorea Electrophoresis. Potyacrylamide gel etectrophoresis using 10% slab gels was carried out at 4 ~ with 40 mA constant current Step Total Total Yield Purl- Specific for approximately 4 h. Tris/glycine (46 mM/35 mM), pH 8.0 activ- pro- fica- activity (_+ 1% SDS) was used as the electrode buffer with bromophenol ity tein tion blue as the tracker dye. Duplicate non-SDS gels were stained (units) (mg) (%) (-fold) (units/rag for protein using the silver stain of Switzer et al. (1979) or protein) for alanopine dehydrogenase activity. To detect enzyme activ- ity, gels were incubated in the dark at room temperature for Crude 340 1,728 - - 0.19 30 min in a mixture of 50 mM Tris-HC1 buffer, pH 8.5 (8 ml), homogenate 50 mM meso-alanopine (2 ml), 10 mg/ml NAD + (2 ml) and I mg/ml nitroblue tetrazolium (5 ml) followed by visualization Ammonium 238 216 70 6 1.10 of the enzyme band by the addition of 0.5 ml 1 mg/ml phen- sulphate azine methosulfate. For SDS gels, protein samples were prein- Sephadex 200 6.9 59 153 29.0 cubated in 1% SDS for at least 2 h at 37 ~ then run and G-100 stained for protein as described above. For the determination of subunit molecular weight using SDS electrophoresis, a plot Chromato- 167 1.9 49 463 88.0 of R I versus molecular weight was constructed using the follow- focusing ing standard proteins: trypsinogen (24,000), ovalbumin (45,000) Sephadex 116 0.48 34 1,263 240.0 and bovine serum albumin (66,000). G-100 Isoelectrofocusing. Isoelectrofocusing was performed by the method of Vesterberg (1971) using an LKB 8101 column Table 2. Substrate specificity of Littorina littorea alanopine de- (110 ml) and a pH gradient of 4.0 to 6.0. Crude enzyme super- hydrogenase. Assay conditions were 50 mM imidaz01e buffer, natant was run at 400 V for 14 h at 4 ~ The column was pH 7.5 with constant co-substrate concentrations, 1.3 mM py- then drained and 1 ml fractions collected and assayed for alano- ruvate, 130 mM g-alanine and 0.1 mM NADH. Relative veloci- pine dehydrogenase activity. ties were measured at saturating levels of each of the amino or keto acids and are expressed relative to the apparent V,~ax Protein Assay. Protein concentration was measured by the with L-alanine and pyruvate as substrates method of Bradford (1976) using the Bio-Rad prepared reagent [ and bovine gamma globulin as the standard. Substrate Apparent K,, Relative (mM) velocity

L-Alanine 47 100 Results Glycine 1,267 37 i L-c~-Aminobutyrate 67 33 Foot Muscle Enzyme Activities L-Serine 220 13 5-Cysteine 35 4 L. littorea foot muscle contains two cytoplasmic Pyruvate 0.55 100 dehydrogenases acting at the pyruvate branch- Oxaloacetate 5.7 94 point: alanopine dehydrogenase (11.1 units/g wet c~-Ketobutyrate 9.4 37 weight) and D-lactate specific lactate dehydroge- Glyoxylate 10.0 10 nase (12.2 units/g wet weight). Octopine dehy- drogenase is not present. SDS gel electrophoresis showed a subunit size of 42,400. Purification and Physical Properties Isoelectrofocusing showed an isoelectric ]point The purification of alanopine dehydrogenase is of 5.6 • 0.02 (n = 3). summarized in Table 1. The enzyme was purified In all preparations only a single band Qf alano- about 1,300-fold to a final specific activity of 240 pine dehydrogenase activity was seen on either units/mg protein and was judged to be homoge- SDS or non-SDS gels stained for proteio or for neous by SDS polyacrylamide gel electrophoresis. enzyme activity. Isoelectrofocusing of the crude en- The molecular weight of L. littorea alanopine zyme preparation showed only a single peak of dehydrogenase, determined by gel filtration, was alanopiue dehydrogenase activity. There ]was no found to be 42,200 +_ 500 (n = 4) similar to that re- evidence, therefore, for multiple molecular forms ported for oyster muscle alanopine dehydrogenase of alanopine dehydrogenase in foot muscl4. (47,000) (Fields and Hochachka 1981) and for oc- topine dehydrogenases (38,000 to 43,000) from Substrate Specificity various sources (Fields et al. 1976; Olomucki et al. The substrate specificity of purified ADHi was as- 1972). Like other imino acid dehydrogenases, L. sessed in terms of apparent K,, values and relative littorea alanopine dehydrogenase was a monomer; velocities (Table 2). L-Alanine and pyruvate were 60 W.C. Plaxton and K.B. Storey: Alanopine Dehydrogenasein Littorina Foot Muscle

0 ~100 20 ~, ,9o 80 15 ,~ u u o o 60

10 Q; > ._> 40 .m o rr 2c

I ~ i i I ! 5 7 8 9 10 pH Fig. 1. pH profiles for L. littorea alanopine dehydrogenase in the forward and reverse directions. Conditions are: o, 1 mM pyruvate, 100 mM L-alanine, 0.1 rnM NADH, 50 mM imidazole buffer (saturating conditions at pH 6.5); o, 1.3 mM pyruvate, 130 mM L-alanine, 0.1 mM NADH, 50 mM imidazole buffer (saturating conditions at pH 7.5); i, 10 mM meso-alanopine, 2 mM NAD +, 50 mM Tris-HC1 buffer; and n, 50 mM meso-alanopine, 2 mM NAD +, 50 mM TAPS (N-tris-(hydroxymethyl)methyl-3-aminopro- panesulfonic acid) buffer. Results are expressed relative to Vm,x for the forward direction which is set at 100 the preferred substrates of the enzyme. Low activi- at pH 7.5) but this is shifted to about 6.5 at lower ty was found with four alternative amino acid sub- substrate concentrations (100 mM L-alanine, strates while three alternative keto acid substrates 1 mM pyruvate, 0.1 mM NADH: saturating sub- were found. Relative activity with oxaloacetate as strate concentrations at pH6.5). This effect an alternative substrate was high but the apparent appears to be related to the pyruvate and alanine K,, for this keto acid was too great to suggest a substrate inhibition characteristics of the enzyme. physiological role for this substrate. No enzyme In the reverse direction, the pH optimum was 9.2 activity was observed with other L-amino acids in- at 50 mM meso-alanopine but dropped to 8.5 at cluding arginine, aspartate, glutamate, histidine, lower (10 raM) levels of meso-alanopine. The ratio isoleucine, leucine, methionine, phenylalanine, of the maximal activities in the forward (at pH 7.5) proline, taurine, tyrosine, asparagine, glutamine, versus the reverse (at pH 9.2) directions was about lysine, threonine, tryptophan or valine, with D-ala- 5:1. nine, with NH 4, or with hydroxypyruvate, 0~-ke- toglutarate, or 0~-ketovalerate. In the reverse direc- Kinetic Constants tion, activity was observed with meso-alanopine only. No activity was apparent with up to 30 mM L. littorea alanopine dehydrogenase followed Mi- D-strombine (2-methyliminodiacetic acid) or chaelis-Menten saturation kinetics for all sub- 200 mM iminodiacetic acid. Similarily, gels stained strates. Kinetic constants for alanopine dehydroge- for alanopine dehydrogenase activity showed no nase are given in Table 3. Due to the marked effect band of enzyme activity when D-strombine was of pH on the Km'S for pyruvate and L-alanine and substituted for meso-alanopine. The enzyme was the importance of intracellular pH in regulating specific for NAD(H) showing no activity in the anaerobic metabolism in marine , the presence of NADP(H). kinetics of the enzyme in the forward direction were examined at two pH's 6.5 and 7.5. The appar- pH Effects ent Km'S for pyruvate and L-alanine were strongly influenced by co-substrate concentration (L-ala- pH profiles of periwinkle muscle alanopine dehy- nine or pyruvate), the apparent K,, for either sub- drogenase are shown in Fig. 1. In the forward, strate being reduced with increasing co-substrate alanopine-producing direction, the enzyme has a concentrations. This effect is well known for octo- pH optimum of 7.5 at high substrate concentra- pine dehydrogenase from a variety of sources tions (130 mM L-alanine, 1.3 mM pyruvate, (Thoai et al. 1969; Fields et al. 1976; Storey and 0.1 mM NADH: saturating substrate conditions Storey 1979b). Table 3 shows the absolute K,,'s W.C. Plaxton and K.B. Storey: Alanopine Dehydrogenase in Littorina Foot Muscle 61

Table 3. Michaelis constants for Littorina littorea alanopine gigas (Fields and Hochachka 1981) muscle alano- dehydrogenase at two pH's. Kinetic constants were derived as pine dehydrogenase showing substrate inhibition outlined in Materials and Methods from analyses of three sepa- by pyruvate. Substrate inhibition at high pyruvate rate preparations of the purified enzyme. Values are means+ s.e.m. Assays were perfomaed at 23 ~ in 50 mM imidazole levels decreased, however, as L-alanine concentra- buffer with constant co-substrate concentrations, where appro- tions were lowered to subsaturating levels(Fig. 2). priate, of 1.3raM pyruvate, 130ram L-alanine, 0.1 mM Comparable effects were found when L-alanine NADH, 30 mM meso-alanopine, and 2 mM NAD + at pH 7.5 substrate inhibition was examined. At saturating and 1.0mM pyruvate, 100raM L-alanine, 0.1 mM NADH, concentrations of pyruvate, substrate inhibition by 15 mM meso-alanopine, and 2 mM NAD + at pH 6.5 L-alanine was apparent above 100 mM at pH 6.5 Substrate pH 6.5 pH 7.5 and above 130 mM at pH 7.5. The apparent Is0's were 450 mM at pH 6.5 and 550 mM ati pH 7.5. Absolute K,, (mM) At subsaturating pyruvate concentration, however, L-Alanine 14.9 _+0.85 23.8 _+0.52 L-alanine substrate inhibition was lost. Pyruvate 0.17 + 0.02 0.26_+ 0.01

Apparent K,~ (raM) Metabolite and Salt Effects NADH 0.009 __ 0.0001 0,009 _+ 0.000J Meso-Alanopine 6.5 _+0.59 16.9 _+1.73 A variety of metabolites was tested for effects on NAD + 0.15 _+0.03 0.18 _+0.003 alanopine dehydrogenase activity in the forward direction at subsaturating substrate concentrations (50 mM imidazole buffer, pH 7.5, 0.5 mM pyru- for pyruvate and L-alanine. K,,'s for both sub- vate, 40 mM L-alanine, and 0.02 mM NADH). The strates were influenced by pH, decreasing by following compounds (at 10 raM) had nO effects 35-37% when pH was lowered from 7.5 to 6.5. on alanopine dehydrogenase activity: glucose-6-P, During anoxia, then, when intracellular pH drops fructose-6-P, phosphoenolpyruvate, ~-lactate, due to an accumulation of acidic end products, malate, fumarate, succinate, arginine phosphate, CO2 and succinate, production of alanopine as the carnitine, acetyl-CoA and the L-amino acids, product of anaerobic glycolysis could be enhanced proline, histidine, methionine and tyrosine. The by this pH effect on . The apparent pyruvate analogue, oxamate (10 raM), the glyco- K,, for NADH was not affected by pH nor was lytic intermediates, fructose-l,6-biphosphate, 3- the apparent Km for NAD + in the reverse direc- phosphoglycerate and dihydroxyacetone-P (all tion. However, the apparent K,, for meso-alano- 10 mM) and the Krebs cycle intermediate,, citrate pine, like those for pyruvate and L-alanine, was and 0~-ketoglutarate (both 10 raM), all produced strongly dependent on pH. While enzyme activity 10-25% inhibition of alanopine dehydrogenase ac- in the reverse direction was highest at pH 8.5, the tivity. Other L-amino acids, arginine, glutamate, K m at this pH was also very high (50 mM) but lysine (all 100mM), leucine, isoleucine (both K,,'s fell with decreasing pH, reaching 6.5 mM at 30 raM) and tryptophan (3 raM) inhibited enzyme pH 6.5. activity by 25-40% compared to controls How- ever, the effects of these inhibitors were not further investigated due to the non-physiological concen- Pyruvate and L-Alanine Substrate Inhibition trations needed to produce the observed inhibitory The effect of pyruvate concentration on the activ- effects. ity of alanopine dehydrogenase is shown in Fig. 2. ATP, ADP, NAD +, meso-alanopine ~, and L- At saturating concentrations of L-alanine, pyru- lactate were inhibitors of alanopine dehydiogenase vate substrate inhibition was apparent at pyruvate (Table 4). Product inhibition by NAD + w]as com- concentrations above 1.0 mM at pH 6.5 and above petitive with respect to NADH while mesO-alano- 1.3 mM at pH 7.5. An I5o of about 8 mM pyruvate pine was a competitive inhibitor with respect to was observed at both pH's, similar to Iso'S of about both pyruvate and L-alanine, the Ki for al[anopine 10 mM reported for pyruvate substrate inhibition being the same (35 raM) with respect to either sub- of H type lactate dehydrogenases and of the brain strate. L-Lactate inhibition is probably a ~esult of specific isozyme of octopine dehydrogenase (Long the structural similarity between L-lactatel and the and Kaplan 1973 ; Eichner and Kaplan 1977; substrates, pyruvate and L-alanine. Inhibition by , I Storey and Storey 1979 a). Muscle forms of lactate L-lactate has no physiological relevance, however, dehydrogenase and octopine dehydrogenase typi- as L. littorea tissues contain a o-lactate dehydroge- cally do not show pyruvate substrate inhibition. nase. The adenylates, ATP and ADP, as' well as However, muscle alanopine dehydrogenases differ their Mg 2 + complexes (ratio 1" 1 Mg: ad,enylate) in this property, both L. littorea and Crassostrea were competitive inhibitors with respect to 62 W.C. Plaxton and K.B. Storey: Alanopine Dehydrogenase in Littorina Foot Muscle

100

"(~ 80

6o

o 40

20

i | i 1 2 4 6 8 1'0 [Pyruvate] mM ;~176f

Fig. 2. Effect of pyruvate substrate concentration ,oE on reaction velocity for alanopine dehydrogenase at saturating and subsaturating, L-alanine concentrations and two pH's. Assays were performed at 23 ~ with 0.1 mM NADH and 50 mM imidazole buffer. Top panel: pH 6.5, alanine concentrations: % 15 mM; zx, 30 mM; o, 100 raM. Bottom panel: pH 7.5, alanine concentrations: [], 30 raM; % 50 mM; o, 130 mM

[Pyruvctte] mM

Table 4. Inhibitor effects on Littorina littorea alanopine dehy- NADH. ATP effects probably occur within the drogenase. Inhibitor constants were determined from Dixon physiological range of ATP concentrations in vivo plots with type of inhibition evaluated by double reciprocal plots. Assay conditions were 50 mM imidazole buffer, pH 7.5 although ATP inhibition of dehydrogenases is with constant concentrations of co-substrates, 1.3 mM pyru- probably due to structural rather than regulatory vate, 130raM L-alanine and 0.1 mM NADH. Values are effects, reflecting the presence of an adenine nucle- means _+ s.e.m, for n=3 determinations on separate prepara- otide domain in the coenzyme tions of purified enzyme. Mg 2 + : adenylate ratio was 1 : 1 (McPherson 1970). To examine interactions between inhibitors and Inhibitor Apparent Ki Inhibition pH, inhibitor effects (Iso) were quantitated at Type With pH 6.5 and 7.5 using sub-saturating substrate lev- respect to els (Table 5). The inhibitory effects of ATP, ADP and NAD § on alanopine dehydrogenase activity NAD + 0.16--+0.012 Competitive NADH decreased (I5o increased) at pH 6.5 compared to ATP 5.6 + 0.25 Competitive NADH pH 7.5 while inhibition by meso-alanopine, D- Mg-ATP 3.0 +0.32 Competitive NADH strombine and L-lactate showed the opposite re- ADP 1.7 -+0.25 Competitive NADH Mg.ADP 4.2 -+0.32 Competitive NADH sponse. Succinate, which had no effect on enzyme Meso- 35.0 -+0.35 Competitive L-alanine and activity at pH 7.5, was inhibitory, at high concen- Alanopine pyruvate trations, at pH 6.5. L-Lactate 3.2 _+ 0.12 Competitive L-alanine Monovalent and divalent ions had inhibitory 5.0 +0.21 Mixed Pyruvate effects on alanopine dehydrogenase activity. Inhi- Competitive bition by 200 mM salt was 16, 23, 33 and 38% W.C. Plaxton and K.B. Storey: Alanopine Dehydrogenase in Littorina Foot Muscle 63

Table 5. Inhibitor effects on Littorina littorea alanopine dehy- ity with glycine. This enzyme is exemplified by drogenase at two pH's. Inhibition constants (Iso) were deter- alanopine dehydrogenase from L. littorea and also mined by the method of Job et al. (1978). Assays were per- occurs in the tissues of another gastropod, the formed in 50 mM imidazole buffer at constant subsaturating substrate conditions: 0.2raM pyruvate, 20raM L-alanine, , Busycotypus canaliculaturn (Plaxton and 0.025 mM NADH at pH 6.5 and 0.25 mM pyruvate, 25 mM Storey 1982). The other enzyme, which is now L-alanine, 0.025 mM NADH at pH 7.5. Values are means of called strombine dehydrogenase by most authors n : 2 determination with variation _+ 10% (de Zwaan and Zurburg 1981 ; Dando et gl. 198/; Inhibitor Iso (mM) Dando 1981) shows glycine activity >_ L-alanine ac- tivity. The enzyme from oyster adductori muscle, pH 6.5 pH 7.5 called alanopine dehydrogenase by Fields and Ho- chachka (198/), is actually a good examplle of this NAD + 0.50 0.38 enzyme type. Both of these occ~tr in the ATP 2.50 /.50 ADP 3.65 2.50 tissues of Mytilus edulis where they are ti~ sue spe- Meso-Alanopine 9.5 25.0 cific and are electrophoretically separable (Dando D-Strombine 14.5 6/.0 et al. 1981; Dando /981). Alanopine dellydroge- L-Lactate 0.90 3.15 nase occurs in many of the soft tissues of ~ f. edulis Succinate 19.0 No inhibition while strombine dehydrogenase is restricted to muscle tissues which have high intracelluli~r levels of glycine as the major free amino acid. for (NH4)2SO4, NH4C1, KC1 and NaC1, respec- Substrate specificities and inhibitori effects tively, at pH 7.5 and subsaturating substrate con- provide information about the substratd site of centrations (0.4mM pyruvate, 40 mM alanine, alanopine dehydrogenase. Carbon chain 1)ngth is important in determining substrate utilization. Cz 0.03 mM NADH). Mg 2+ ions were also inhibito- I ry; at 50 mM salt, inhibition was 30 and 45% in (glycine, glyoxylate) and C4 (L-0~-amlnobutyrate, 0~-ketobutyrate) substrates were utilized t,ut only the presence of MgSO 4 and MgC12, respectively. at reduced rates and with high apparent K~ values compared to pyruvate and L-alanine. Alt:rations Discussion to a C 3 substrate (the hydroxyl groups, ,f serine and hydroxypyruvate, the sulfhydryl grou of cys- Alanopine dehydrogenase, purified from L. lit- teine) also reduced enzyme activity indicating the torea, differs most strongly from purified oyster importance of the methyl groups of L-Mar Line and muscle alanopine dehydrogenase in the substrate pyruvate in substrate binding. Compo~mds of specificities of the enzyme. L. littorea alanopine chain length greater than C4 or with subs itutions dehydrogenase is very specific for L-alanine and on the C4 chain (threonine, methionine, valine, pyruvate as substrates; maximal activities with leucine, isoleucine, ~-ketovalerate, ~-keioglutar- other amino acids were no more than 1/3 of that ate) were inactive. The enzyme was also .inactive found with L-alanine while alternate keto acids with D-alanine. D-lactate, the product o: the D- showed high (and unphysiological) apparent K,, lactate dehydrogenase of L. littorea, had ~o effect values and/or low maximal velodties. By contrast, on alanopine dehydrogenase but L-lactat~was in- oyster alanopine dehydrogenase showed virtually hibitory with respect to both L-alanine arM pyru- equal maximal activities with L-alanine, glycine, L- vate. This suggests the importance of the L ~onfigu- serine or L-0~-aminobutyrate as well as equal appar- ration at the substrate site. ent Km values for L-alanine and glycine (Fields and The kinetic properties of L. tittorea al~mopine Hochachka 198/). The amino acid substrate speci- dehydrogenase can suggest a physiological~ole for ficities of imino acid dehydrogenases may be, in the enzyme in redox regulation during am erobio- part, related to the predominant free amino acids sis. Two properties are particularily persuasive. available in vivo in each ; in L. littorea ala- Firstly, alanopine dehydrogenase shows a depen- nine is one of the most abundant free amino acids dence of the apparent K,, for pyruvate of L alanine in foot muscle (at /8 gmol/g wet weight in animals on co-substrate (L-alanine or pyruvate) coJlcentra- acclimated to 100% seawater; Hoyaux et al. 1976). tion, rising levels of either the amino or k,,'to acid However, it is now becoming apparent, that of co-substrate decreasing the apparent K m 10r keto imino acid dehydrogenases using L-alanine and/or or amino acid, respectively. This effect is well glycine as substrates, two distinct enzymes can be known for octopine dehydrogenase and it 1)as been identified. One enzyme has a high specificity for suggested that this property is a key factor in- L-alanine as its substrate and shows very low activ- fluencing enzyme activity in vivo and pr+moting 64 W.C. Plaxton and K.B. Storey: Alanopine Dehydrogenase in Littorina Foot Muscle the synthesis of octopine as the product of glyco- gesting distinct roles for the two enzymes. Lactate lytic muscle work (Fields et al. 1976). In L. littorea, dehydrogenase may function during muscle work. where alanine is a major end product of anaerobic metabolism (Wieser 1980), the activation of glyco- Acknowledgements. The authors are grateful to Dr. P.R. Dando lysis during anoxia could lead to both alanine and and Dr. J. Tempe for their gifts of meso-alanopine and D- strombine. Michael Cooke provided technical assistance with alanopine accumulation as end products. Indeed, isoelectrofocusing. We thank Dr. J.H.A. Fields for helpful dis- studies in the oyster have shown that alanopine/ cussions in the preparation of the manuscript. Support was strombine are substantial products of anaerobiosis provided by an N.S.E.R.C. operating grant and by N.R.C. in ventricle accounting for 35% of 14C-glucose ca- contract #OSU80-00218. tabolized during one hour of anoxia (Collicutt and Hochachka 1977). Secondly, L. littorea alanopine References dehydrogenase is strongly affected by pH. The K,,'s for both pyruvate and L-alanine decrease with Baldwin J, England WR (1980) A comparison of anaerobic decreasing pH, an effect which would promote energy metabolism in mantle and tentacle muscle of the alanopine synthesis during anoxia when intracellu- blue-ringed , Hapalochlaena maculosa during swim- ming. Aust J Zool 28:407-412 lar pH declines. For oyster muscle alanopine dehy- Bradford MM (1976) A rapid and sensitive method for the drogenase, this effect of pH of K,,'s for alanine quantitation of microgram quantities of protein utilizing and pyruvate was not found although pH had a the principle of protein-dye binding. Anal Biochem slight effect on K m for NADH (Fields and Ho- 72: 248-254 chachka 1981). Collicutt JM, Hochachka PW (1977) The anaerobic oyster heart. Coupling of glucose and aspartate fermentation. J Alanopine or strombine production as prod- Comp Physiol 115:147-157 ucts of anaerobic metabolism in marine molluscs Cornish-Bowden A (1979) Fundamentals of enzyme kinetics. has now been reported by several authors (de Butterworths, London, pp 109-116 Zwaan and Zurburg 1981; Fields 1977; Collicutt Dando PR (1981) Strombine (N-(carboxymethyl)-D-alanine) dehydrogenase and alanopine (meso-N-(1-carboxyethyl)-al- and Hochachka 1977). In Mytilus edulis, accumu- anine) dehydrogenase from the Mytilus edulis L. lation of strombine was greatest during the first Biochem Soc Trans 9 : 297-298 six hours of anoxia (de Zwaan and Zurburg 1981) Dando PR, Storey KB, Hochachka PW, Storey JM (1981) Mul- suggesting that the activation of anaerobic glycoly- tiple dehydrogenases in marine molluscs: electrophoretic sis to produce products such as alanine and alano- analysis of alanopine dehydrogenase, strombine dehydroge- nase, octopine dehydrogenase and lactate dehydrogenase. pine/strombine is the primary response of these Mar Biol Lett 2:249 257 animals to anoxia. Long term anoxia, however, Eichner R, Kaplan NO (1977) Catalytic properties of lactate is met by a secondary response in which glycolytic dehydrogenase in Homarus americanus. Arch Biochem carbon flow is channeled into the production of Biophys 181:501-507 Ellington WR (1979) Evidence for a broadly-specific, amino succinate and volatile fatty acids. Thus the primary acid requiring dehydrogenase at the pyruvate branchpoint function of alanopine dehydrogenase in vivo in sea anemones. J Exp Zool 209:151 159 appears to be in redox regulation of glycolysis dur- Ellington WR (1981) Energy metabolism during hypoxia in ing the initial hours of anaerobic function. the isolated, perfused ventricle of the whelk, con- Imino acid dehydrogenases can also function, trarium Conrad. J Comp Physiol 142:457-464 Fields JHA (1976) A dehydrogenase requiring alanine and py- like lactate dehydrogenase, in providing glycolytic ruvate as substrates from oyster adductor muscle. Fed Proc redox balance during 'burst' muscle work when 35:1687 supplies are insufficient to meet energy Fields JHA (1977) Anaerobic metabolism in the , Clino- demand by contracting muscle. This function cardium nuttali. Am Zool 17:943 Fields JHA, Baldwin J, Hochachka PW (1976) On the role appears to be the primary function of octopine of octopine dehydrogenase in cephalopod mantle muscle dehydrogenase in the species in which the enzyme metabolism. Can J Zool 54:871-878 occurs (Grieshaber and G/ide 1976; Storey and Fields JHA, Hochachka PW (1981) Purification and properties Storey 1979b) and must also apply to alanopine of alanopine dehydrogenase from the adductor muscle of or strombine dehydrogenases in certain species. In the oyster, Crassostrea gigas. Eur J Biochem 114: 615-621 Grieshaber M, G/ide G (1976) The biological role of octopine the Oyster, for example, neither lactate dehydroge- in the , Loligo vulgaris. J Comp Physiol 108:225-232 nase nor octopine dehydrogenase are present, so Hoyaux J, Gilles R, Jeuniaux C (1976) Osmoregulation in mol- alanopine dehydrogenase must play a dual role in luscs of the . Comp Biochem Physiol [A] the response to both muscle work and anoxia stress 53 : 361-365 in vivo. In L. littorea foot muscle, however, both Job D, Cochet C, Dhien A, Chambaz EM (1978) A rapid meth- od for screening inhibitor effects: Determination of I5o and alanopine dehydrogenase and lactate dehydroge- its standard deviation. Anal Biochem 84:68-77 nase occur in approximately equal activities sug- Long G, Kaplan NO (1973) Lactate dehydrogenase from the W.C. Plaxton and K.B. Storey: Alanopine Dehydrogenase in Littorina Foot Muscle 65

horseshoe crab, Limulus polyphemus. Arch Biochem Storey KB, Storey JM (1979b) Octopine metabolism in the Biophys 154: 69(~710 , Sepia officinalis: octopine production by muscle McPherson A (1970) Interaction of lactate dehydrogenase with and its role as an anaerobic substrate for non-muscular tis- its coenzyme, nicotinamide-adenine dinucleotide. J Mol Biol sues. J Comp Physiol 131:311-319 51 : 39-46 Switzer RC, Merril CR, Shifrin S (1979) A highly sensitive Olomucki A, Huc C, Lefebure F, Thoai N van (1972) Octopine stain for detecting proteins and peptides in polyacrylamide dehydrogenase. Evidence for a single chain structure. Eur gels. Anal Biochem 98:23/ 237 J Biochem 28 : 261-268 Thoai N van, Huc C, Pho DB, Olomucki A (1969) Octopine Plaxton WC, Storey KB (1982) Tissue specific isozymes of alan- d~shydrog6nase. Purification et propri~t~s catalytiques. opine dehydrogenase in the channeled whelk, Busycotypus Biochim Biophys Acta 191:46-57 canaliculatum. Can J Zool (in press) Vesterberg O (1971) Isoelectrofocusing of proteins. In: Colow- Regnouf F, Thoai N van (1970) Octopine and lactate dehy- ick SP, Kaplan NO (eds) Methods in enzymology. Academ- drogenases in mollusc muscles. Comp Biochem Physiol ic Press, New York, pp 389412 32:411-416 Wieser W (1980) Metabolic end products in three species of Storey KB, Storey JM (1979a) Kinetic characterization of tis- marine gastropods. J Mar Biol Ass UK 60:/75-180 sue-specific isozymes of octopine dehydrogenase from Zwaan A de, Zurburg W (198/) The formation of strombine mantle muscle and brain of Sepia offieinalis. Eur J Biochem in the adductor muscle of the sea mussel, Mytilus edulis 93 : 545-552 L. Mar Biol Lett 2:17%192