tissue‐specific alanopine dehydrogenase and strombine dehydrogenase from the sea mouse, aphrodite...

THE JOURNAL OF EXPERIMENTAL ZOOLOGY 225:369-378 (1983)

Tissue-Specific Alanopine Dehydrogenase and Strombine Dehydrogenase From the Sea Mouse, Aphrodife aculeata (Po I yc h aet a)

KENNETH B. STOREY Institute of Biochemistry, Carleton Uniuersity, Ottawa, Ontario, Canada K1S 5B6

ABSTRACT Each tissue of the sea mouse, Aphrodite aculeata, contains a single cytoplasmic dehydrogenase acting at the pyruvate branchpoint: Alano- pine dehydrogenase (ADH) occurs in longitudinal muscle, nerve, and elytra, and strombine dehydrogenase (SDH) characterizes pharynx and intestine. Lac- tate and octopine dehydrogenases were not detected in any tissue. ADH from muscle and SDH from pharynx were partially purified. The two enzymes had the same molecular weight, 44,000 k 4,000 but differed in isolelectric point. Although similar in keto acid specificity, the enzymes differed strongly in amino acid specificity. Pharynx SDH showed a very high specificity for glycine, all other alternative amino acids showing very high apparent K,s andlor low Vrnaxs. Muscle ADH, however, displayed high velocities and relatively low apparent K,s with L-alanine, L-serine, L-threonine, and L-cysteine. Amino acid analysis of the tissues showed that serine and threonine contents were high enough in muscle to suggest that these amino acids might be alternative substrates to alanine in vivo; only glycine, present a t 382 pmol/g in pharynx, is likely to be a physiological substrate for SDH, however. Absolute K,s for L- alanine, glycine, and pyruvate were 2.75 k 0.03,285 f 6, and 0.2 f 0.006 for muscle ADH and 130 k 2.2,26 f 1 and 0.15 & 0.005 for pharynx SDH. In the reverse direction, apparent K,s for meso-alanopine, D-strombine, and NAD, at pH 9.5, were 1.68 f 0.06, 87.0 (n = 11, and 0.75 f 0.03 for ADH and 6.75 f 0.20, 7.57 & 0.07, and 0.54 f 0.02 for SDH. The enzymes also differed strongly in effects of inhibitors. Meso-alanopine, L-lactate, and D-lactate were strong inhibitors of the forward reaction for muscle ADH. Pharynx SDH, however, was strongly inhibited by meso-alanopine and iminodiacetic acid but showed only weak inhibition by D- or L-lactate or by its product, D-strombine.

Alanopine dehydrogenase and strombine dehydrogenase activities, catalyzing the reactions,

L-alanine + pyruvate + NADH + H+ meso-alanopine + NAD* + H2O

glycine + pyruvate + NADH + H+ D-strombine + NAD' + H2O

H&-CH-COOH CH&OOH I I

NH I I NH

H,C-CH-COOH HsC-CH-COOH

Alanopine Strornbine

respectively, have been identified in the tissues of many marine invertebrates in recent years.

The enzyme activities were first reported in the oyster (Fields, '76) and subsequently have been found in many bivalve and gastropod species (Fields, '77; Dando et al., '81; de Zwaan and Zurburg, '81) as well as in sponges (Barrett and Butterworth, '811, sea anemones (Ellington, '79), and polychaete worms (de Zwaan, personal communication). Kinetic properties of the purified enzymes from the oyster (Fields and Ho- chachka, '81) and from the periwinkle and whelk (Plaxton and Storey, '82a, '83) have been reported.

The enzymes are highly variable in their amino acid specificities; a single enzyme may utilize not only L-alanine and glycine but also other amino acids including L-serine, L- threonine, L-valine, and L-cysteine. A gly-

0 1983 ALAN R. LISS. INC.

370 K.B. STOREY

cine-specific strombine dehydrogenase occurs in the sponge, Halichondria panicea (Barrett and Butterworth, %l), and enzymes with high specificities for L-alanine occur in gastropod species (Plaxton and Storey, '82a, b). However, the muscle enzymes from three bivalve mollusks show similar rates and similar apparent K,s for both glycine and L- alanine (Fields and Hochachka, '81; Dando, '81; Storey et al., '82). Tissue-specific differences in enzyme amino acid specificity also occur (Dando et al., '81); while the adductor muscle enzyme from Mytilus edulis shows a similar affinity for both L-alanine and glycine, the foot muscle form shows a strong preference for L-alanine (Dando, '81).

The present study provides the first exam- ination of these imino acid dehydrogenases in a polychaete worm. Lactate dehydrogenase activities are low in many polychaete species (Scheid and Awapara, '72) and octopine dehydrogenase appears to be absent in the class (de Zwaan, personal communication); alanopine or strombine dehydrogenases, therefore, provide the major (or sole) means for cytoplasmic redox balance during glycolytic energy production related to muscular work or anaerobiosis. The present study examines the probable roles of alanopine and strombine dehydrogenases in Aphrodite aculeata, and is additionally interesting owing to the presence of distinct tissue-specific forms of the enzymes. Longitudinal muscle from the sea mouse displayed an enzyme with a high affinity for L-alanine and low affinity for glycine, an alanopine dehydrogenase (ADH). Pharynx muscle, by contrast, contains an enzyme with a strong specificity for glycine, a strombine dehydrogenase (SDH).

MATERIALS AND METHODS Animals and chemicals

Specimens of the sea mouse, A. aculeata, were collected at the Marine Biological Lab- oratory, Plymouth, and were stored frozen at -80°C until use. All biochemicals were from Sigma Chemical Co.; Polybuffer 74, PBE 94 chromatofocusing exchanger, Sephadex G- 100, and DEAE-Sephadex were from Phar- macia Fine Chemicals. D-Strombine and meso-alanopine were synthesized as outlined by Tempe ('82).

Tissue enzyme activity measurements Tissues were dissected out of thawed speci-

mens, blotted, weighed, and homogenized (1:lO wlv for nerve, 1:5 w/v for others) in 50 mM imidazole-HC1 buffer, pH 7.0, containing 15 mM 2-mercaptoethanol using a Polytron

PT 10-35 homogenizer. Homogenates were centrifuged at 27,OOOg for 30 min at 4°C. Supernatants were removed, dialyzed over- night a t 4°C against 1,000 vol of homogenizing buffer, and then used for the determination of enzyme activities.

Enzyme purification Pharynx muscle and longitudinal muscle

(both dorsal and ventral bands) were dissected out of thawed specimens. Tissues were homogenized in 5 vol (w/v) 25 mM imidazole- HC1 buffer, pH 7.0, containing 20 mM 2-mercaptoethanol and centrifuged as above.

For partial purification of pharynx SDH, crude supernatant was layered onto a chromatofocusing column (30 x 0.9 cm) of PBE 94 exchanger equilibrated in homogenizing buffer. Enzyme was eluted with a pH 7 to 4 gradient of Polybuffer 74. Peak fractions were pooled, readjusted to pH 7, and loaded onto a column of Sephadex G-100 (90 x 1.5 cm). Enzyme was eluted in homogenizing buffer. Peak fractions from this column were pooled and used as the source of enzyme for kinetic studies.

For partial purification of muscle ADH, crude supernatant was loaded onto a column of DEAE-Sephadex (5 x 2 cm) equilibrated in homogenizing buffer. Enzyme was eluted with a linear gradient (100 ml) of 0-250 mM KCl in buffer. Peak fractions (eluting at 120- 150 mM salt) were pooled and applied to a Sephadex G-100 column as above. Peak fractions from this column were pooled and used as the source of enzyme for kinetic studies.

Partially purified enzymes were stable a t 4°C for a t least 1 week. Protein determinations were made by the method of Bradford ('76) using the prepared reagent from Bio- Rad Laboratories and bovine gamma globu- lin as the standard.

Molecular weight determination Molecular weight determinations were per-

formed on a column of Sephadex G-100 (90 x 1.5 cm) using 25 mM imidazole-HC1 buffer, pH 7, containing 20 mM 2-mercaptoethanol as the equilibratiodelution buffer. Molecular weight was determined from a plot of K,, versus log M, for standards: ribonuclease (M, 13,700), chymotrypsinogen (M, 25,000), oval- bumin (M, 45,000), and bovine serum albu- min (M, 67,000).

Enzyme assay and kinetic studies Standard assay conditions for the enzymes

were 50 mM imidazole-HC1, pH 7.0, 0.1 mM NADH, and 200 mM L-alanine for muscle

ADH AND SDH FROM A. ACULEATA 371

TABLE 1. Tissue imino acid dehydrogenase actiuities in the sea mouse, Aphrodite aculeata

Ratio L-alanine Glycine ratio ratio 2 0 0 a l d Enzyme

200mM 600mM 200/600 200mM 600mM 200/600 200gly type

Muscle 47.5 f 4.6 35.7 f 3.6 1.33 30.7 * 4.3 51.2 f 7.4 0.60 1.55 ADH Pharynx 45.3 f 3.6 51.1 f 5.6 0.89 52.3 k 2.0 36.5 f 0.8 1.43 0.87 SDH Intestine 4.6 ? 0.87 5.2 f 1.28 0.89 5.3 f 1.05 5.0 i 0.97 1.06 0.87 SDH Nerve 16.8 f 3.8 15.5 k 3.3 1.08 10.1 f 2.7 19.4 + 4.3 0.52 1.66 ADH Elytra 4.5 f 0.44 3.9 k 0.35 1.15 2.8 k 0.30 5.5 f 0.52 0.50 1.61 ADH Enzyme activities are given as rmol NADH utilizedlminig wet weight k SEM (n = 3) at 23°C. Cosubstrate concentrations were 0.1 mM NADH and 2 mM pyruvate in 50 mM imidazole-HC1 buffer, pH 7.0. Muscle includes the dorsal and ventral longitudinal muscle bands while nerve is the ventral nerve cord

ADH or 200 mM glycine for pharynx SDH in the forward direction. For the reverse direction, conditions were 50 mM Tris-HC1 buffer, pH 9.5, 2 mM NAD, and 10 mM meso-alanopine for ADH or 20 mM D-strombine for SDH. Activity was assayed by monitoring NAD(H) utilization at 340 nm using a Pye Unicam SP 8-100 recording spectrophotome- ter with water-jacketed cell holder. One unit of enzyme activity is defined as the amount of enzyme utilizing 1 pmol NADH per min at 23°C with L-alanine as the substrate for ADH or glycine as the substrate for SDH.

Apparent Kms were determined from di- rect linear (Cornish-Bowden, '79) or Hanes plots. Absolute K,s for alanine, glycine, and pyruvate were derived from secondary plots (S,Nap, versus ,5321, V, being derived from determinations of cosufktrate apparent K,s at four substrate (S2) concentrations (Cor- nish-Bowden, '79). K i s were determined from Dixon plots with the effects of inhibitors evaluated by double reciprocal plots.

Amino acid determination Frozen tissues were ground to a powder

under liquid nitrogen, rapidly weighed, and then homogenized in 5 vol (w/v) ice-cold 50 mg/ml sulphosalicyclic acid using a Polytron homogenizer. Precipitated protein was removed by centrifugation at 27,OOOg for 30 min at 4°C. Amino acids in the supernatant were quantitated using a Beckman 119BL amino acid analyzer.

RESULTS Tissue enzyme levels

L-alanine-dependent and glycine-dependent imino acid dehydrogenase activities were present in all tissues tested in the sea mouse (Table 1). However, neither lactate dehydrogenase nor octopine dehydrogenase (tested at both 1 and 20 mM L-arginine) activities were found in any tissue. Tissue-spe-

cific differences in imino acid dehydrogenase complement were found. Two enzyme types were indicated based on their amino acid substrate saturation curves. Longitudinal muscle, nerve, and elytra showed an enzyme with a high specificity for L-alanine, saturating levels being 100-200 mM with some substrate inhibition at higher alanine concentrations (activity ratio 200/600 mM alanine being 1.08-1.33). The glycine-dependent enzyme activity in these tissues, however, reached a maximum only at 600 mM glycine. Pharynx muscle and intestine showed an enzyme activity that was much more strongly glycine-dependent. Maximal glycine activity was found at 200 mM glycine with higher levels being inhibitory. Alanine activity, however, was not maximal until 600 mM. The two enzyme types have been designated alanopine dehydrogenase (ADH) for the enzyme from muscle, nerve, and elytra, and strombine dehydrogenase (SDH) for the enzyme from pharynx and intestine.

The enzyme activities from longitudinal muscle and pharynx were chosen for further characterization. Extracts from each tissue were run on chromatofocusing columns. Each tissue displayed only a single enzyme form, the muscle form eluting at pH 5.55 and the pharynx form at pH 5.70 (chromatofocusing provides approximate PIS for enzymes) (Fig. 1). Each enzyme retained, after chromatofocusing, the alaninelglycine activity ratio characteristic of the crude enzyme prepara- tion. Polyacrylamide gel electrophoresis (with staining using NAD plus meso-alanopine or D-strombine) also indicated only a single enzyme form in each tissue.

Enzyme purification Muscle ADH and pharynx SDH were par-

tially purified for kinetic study. The muscle enzyme was purified 32-fold to a final specific activity of 90 U/mg protein (Table 2); phar-

372 K.B. STOREY

301 PHARYNX MUSCLE

5.0

5.8

5.6

PH

5.4

5.2

Fig. 1. Chromatofocusing elution profiles for pharynx SDH and muscle ADH from A. aculeata. Enzymes were chromatofocused using a pH 7 to 4 gradient. One-rnilli- liter fractions were collected. Enzyme assay conditions

are as in Table 1 with 200 mM L-alanine (0) or 200 mM glycine (0). Determination of pH of fractions (-=-I shows approximate PIS of 5.7 for pharynx SDH and 5.55 for muscle ADH.

TABLE 2. Purification of alanopine dehydrogenase and strombine dehydrogenase from Aphrodite aculeata

Specific Total Total Purifi- activity

activity protein Yield cation (unitsirng Step (units) (rng) ('70) (fold) protein) Muscle ADH

2.84 Crude homogenate 56.3 19.8 - -

Sephadex G-100 33.8 0.38 90.0 3.1 90.0

Crude homogenate 110.2 64.7 - -

DEAE-Sephadex 37.6 1.3 66.8 10.2 28.9

Pharynx SDH 1.70

Chromatofocusing 67.3 1.01 61.0 40.0 68.0 Sephadex G-100 60.6 0.30 90.0 3.0 204.0

ynx SDH was purified 120-fold to a final specific activity of 204 U/mg protein. The instability of muscle ADH to chromatofocusing prevented the use of this technique for the purification of ADH. These partial puri- fications compare to final specific activities of 240 U/mg and 318-385 U/mg for ADH purified to homogeneity from Littorina l i t te rea and Busycotypus canaliculatum, respectively (Plaxton and Storey, '82a, '83). Both enzyme preparations were tested and found

to be free of enzyme activities (malate dehydrogenase, a-glycerol-P dehydrogenase, alanine aminotransferase, pyruvate kinase) that might interfere with kinetic studies of ADH and SDH.

pH profiles pH optima in the forward direction were

rather broad, pH 6.5-7.0 for muscle ADH and pH 6.0-6.5 for pharynx SDH, in either imidazole or MES buffers. In the reverse direction,


TABLE 3. Substrate specificities of muscle alanopine dehydrogenase and pharynx strombine dehvdroeenase from Auhrodite aculeata

Muscle ADH Pharynx SDH

Amino acids Krn(app) Vmax Km(app) Vmax

L-alanine 12.5 100.0 170.0 101.1 Glycine 430.0 161.5 36.5 100.0 L-serine 48.0 150.8 360.0 88.5 L-valine 70.5 71.5 310.0 11.2 L-threonine 25.5 100.0 48.0 9.3 L-cysteine 12.0 130.8 28.0 19.9 L-a-aminobutyrate 9.0 60.0 457.5 68.3

Pyruvate 0.27 100.0 0.47 100.0 Keto acids

a-Ketobutyrate 2.35 21.0 3.55 24.8 a-Ketovalerate 9.1 25.6 23.8 19.6 Oxaloacetate 0.90 106.6 1.7 106.3 Hydroxypyruvate 2.3 12.8 8.5 29.5 Glyoxylate > 50 - 32.5 23.6

Assay conditions were: 50 mM imidazole buffer, pH 7.0, 0.1 mM NADH, 2 mM pyruvate (for amino acid specificities), and 200 mM L-alanine (for ADH keto acid specificities) or 200 mM glycine (for SDH keto acid specificities). Data are given as apparent K,s and calculated V,,, and are the average of two determinations (variability t 10%) using separate preparations of the partially purified enzymes. K,s are given in mM and V,,,s are given relative to activities with pyruvate + L-alanine (for ADH) or pyruvate + glycine (for SDH) which are set at 100.

both enzymes had pH optima of approxi- mately 9.0 in either Tris or diethanolamine buffers.

Molecular weights Sephadex G-100 chromatography showed

that both enzymes, within experimental er- ror, had the same molecular weight, 44,000 f 4,000. This compares with values of 47,000 for oyster muscle ADH (Fields and Ho- chachka, '81), and 41,000-43,000 for ADHs from periwinkle and whelk (Plaxton and Sto- rey, '82a, '83). Like the enzymes from other species, sea mouse ADH and SDH appear to be monomers.

Substrate specificities The two enzymes showed distinct differ-

ences in their amino acid substrate specificities (Table 3). Seven amino acids were utilized by the enzymes; others, including D-alanine, L-arginine, L-lysine, L-aspartic acid, L-glu- tamic acid, L-proline, L-methionine, L-isoleu- cine, L-tryptophan, L-leucine, L-phenyl- alanine, L-glutamine, and L-ornithine, as well as taurine and ammonium ion were not substrates for the enzymes. Muscle ADH showed a high affinity for L-alanine (apparent K, = 12.5 mM) but a very low affinity for glycine (Km(a p) = 430 mM). The enzyme also had high adnities for L-cysteine and L- a-aminobutyrate and intermediate affinities, coupled with high V,,, activities, for L- serine, L-valine, and L-threonine. Pharynx

SDH, by contrast, was highly specific for glycine. Apparent K,s for L-alanine, L-serine, L-valine, and L-a-aminobutyrate were very high whereas maximal velocities with L- threonine or L-cysteine were very low. Amino acids other than glycine, therefore, appear to be poor substrates for the enzyme.

Both enzymes showed similar keto acid specificities. Oxaloacetate was the best alternative keto acid substrate, showing maximal velocity equal to that of pyruvate and a low apparent K,. Other keto acids showed much lower V,, activities and high apparent K,s. Apparent K, increased with increasing car- bon chain length, pyruvate vs. a-ketobutyrate vs. a-ketovalerate whereas a-keto- glutarate was not utilized by either enzyme. Glyoxylate was a poor substrate for both enzymes.

In the reverse, imino acid oxidizing, direction both enzymes could oxidize meso-alanopine and D-strombine but neither oxidized iminodiacetic acid or D-octopine. Meso-alanopine was the preferred substrate of the muscle enzyme, the activity ratio alanopine/ strombine being 5 1 (at 7 mM imino acid, 2 mM NAD, 50 mM Tris buffer, pH 9.5). The activity ratio, alanopinelstrombine, for the pharynx enzyme, however, was approxi- mately 1:l (at 20 mM imino acid).

Activity ratios for the forward versus reverse reactions under standard assay conditions were 6:l for muscle ADH and 7.4:l for pharynx SDH.

374 K.B. STOREY

TABLE 4. Kinetic constants of the forward reaction for muscle alanopine dehydrogenase and pharynx strombine

dehydrogenase from Aphrodite aculeata

Muscle ADH Pharynx SDH

Km(abs1mM

L-Alanine 2.75 k 0.03 130.0 f 2.2 Glycine 285.0 zk 6.0 26.0 * 1.0 Pyruvate (wrt alanine) 0.200 f 0.006 0.18 f 0.004 Pyruvate (wrt glycine) 0.105 k 0.003 0.15 f 0.005

NADH 8.3 + 0.75 10.5 + 2.0 Krn(app) r M

Data are means k SEM for three determinations on separate preparations of the partially purified enzymes. Absolute K,,s were determined as outlined in Materials and Methods with NADH at 0.1 mM. Apparent K,,s for NADH were determined at 2 mM pyruvate and 200 mM L-alanine (for ADH) or 200 mM glycine (for SDH). All assays were performed in 50 mM imidazole buffer, pH 7.0.

Substrate afinity constants Forward direction, Table 4 shows the Mi-

chaelis constants for substrates of the forward reaction. Muscle ADH showed a very high affinity (absolute K , = 2.75 mM) for L- alanine but a very low affinity for glycine (Kr?(abs) = 285 mM). Absolute K,s for the amino acids showed the opposite trend for pharynx SDH, for glycine being over threefold lower than the Km(abs) for L-alanine. The two enzymes had similar absolute K,s for pyruvate, however, with either alanine or glycine as substrate. Apparent K,s for NADH were also similar for the two enzymes.

Reverse direction. Table 5 shows kinetic constants for the reverse reaction for muscle ADH and pharynx SDH. Muscle ADH had a very high affinity for meso-alanopine but showed an extremely low affinity for D- strombine, the results being consistent with the alaninelglycine utilization pattern of the forward reaction. Pharynx SDH, however, showed similar affinities for both imino acids, and K m ( ~ ~ ~ ) was only slightly lower in the presence of alanopine. Affinities for all substrates (for both enzymes) increased when pH was lowered from 9.5 to 8.0.

Metabolite and salt effects. Metabolite and salt effects on the enzymes were tested at subsaturating substrate (0.8 mM pyruvate, 10 mM L-alanine, or 60 mM glycine) levels. Salts, KC1, NaC1, NH4C1, (NH4)2S04 (all a t 200 mM), MgCl2 (50 mM), CaC12, and LiCl (both 20 mM) had no effect on enzyme activity.

Neither enzyme was affected by the addition of L-arginine, succinate, citrate, cy-gly- cero-P or fructose-6-P (all a t 20 mM), oxamate

TABLE 5. Kinetic constants of the reuerse reaction at two pHs for muscle alanopine dehydrogenase and

pharynx strombine dehydrogenase from Aphrodite aculeata

pH 9.5 pH 8.0

Muscle ADH Meso-alanopine 1.68 f 0.06 1.92 f 0.26 D-strombinea 87.0 > 100 NAD (wrt alanopine) 0.75 ~f: 0.03 1.25 f 0.20

Meso-alanopine 6.75 f 0.20 7.0 f 0.28 D-strombine 7.57 f 0.07 10.1 f 0.38 NAD (wrt strombine) 0.54 f 0.02 0.63 f 0.05 NAD (wrt alanopine) 0.30 f 0.04 0.43 f 0.04

Apparent K, determinations were made using 50 mM Tris-HC1 buffer with cosubstrate concentrations 2 mM NAD, 10 mM meso- alanopine for muscle ADH or 20 mM D-strombine or meso- alanopine for pharynx SDH. Determinations are means f SEM for three determinations on separate preparations of the partially purified enzymes. "Single determination only.

Pharynx SDH

(10 mM), P-enolpyruvate, D-octopine (both 5 mM), ADP, AMP (both 2 mM), or fructose- l,6-P2 (1 mM).

Substrate inhibitors At saturating amino acid concentrations,

both enzymes showed limited substrate inhibition by pyruvate. For pharynx SDH, 50% inhibition was observed at 20 mM pyruvate, and enzyme velocity was reduced by 40% at 30 mM pyruvate for muscle ADH. Pyruvate substrate inhibition was reduced when subsaturating amino acid levels were used; a similar phenomenon was noted by Plaxton and Storey ('82a). Pharynx SDH was inhibited at glycine levels greater than 200 mM with 50% inhibition occurring at 800 mM glycine. L-alanine substrate inhibition of muscle ADH was also apparent (Table 1).


TABLE 6. Inhibitor effects on Aphrodite aculeata alanopine dehydrogenase and strombine dehvdrogenase

Inhibition Inhibitor Apparent Ki Type With respect to

Muscle ADH meso-alanopine 2.0 Competitive L-alanine

1.0 Noncompetitive Pyruvate L-lactate 1.9 Competitive L-alanine

6.0 Mixed competitive Pyruvate D-lactate 4.2 Competitive L-alanine

19.0 Competitive Pyruvate

meso-alanopine 3.4 Mixed competitive Glycine 4.1 Mixed competitive Pyruvate

D-strombine 27.0 Mixed competitive Glycine 44.0 Noncompetitive Pyruvate

Iminodiacetic acid 3.7 Competitive Glycine 3.5 Mixed competitive Pyruvate

Pharynx SDH

Assay conditions were: 50 mM imidazole buffer, pH 7.0, 0.1 mM NADH, cosubstrates at 2 mM pyruvate, 200 mM L- alanine or 200 mM glycine.

Substrate inhibition of the reverse direction by meso-alanopine and D-strombine was strong. ADH activity was inhibited by 50% at 20 mM meso-alanopine at pH 9.5, or at 40 mM meso-alanopine at pH 8.0.

Other inhibitors Both enzymes showed product inhibition of

the forward reaction by NAD with a 50% decrease in enzyme activity a t 2-3 mM NAD. ATP also inhibited enzyme activity, 10 mM ATP inhibiting ADH by 33% and SDH by 50%. ATP inhibited with respect to NADH as occurs for the enzyme from other sources (Fields and Hochachka, '81; Plaxton and Sto- rey, '82a).

Meso-alanopine was a strong product inhibitor of both enzymes, but D-strombine had no effect on muscle ADH and only slightly inhibited pharynx SDH. D- and L-lactate were strong inhibitors of muscle ADH, but showed only weak effects (less than 10% inhibition at 20 mM) on pharynx SDH. Iminodiacetic acid strongly inhibited SDH but not ADH. Both enzymes were inhibited by acetate.

Apparent inhibition constants (Ki) are shown in Table 6. With the exception of iminodiacetic acid inhibition of SDH, inhibitions were in all cases stronger with respect to the amino acid substrate than with respect to pyruvate. L-lactate inhibition of ADH was stronger than was D-lactate inhibition; this may be related to the stereochemistry of the active site. Surprisingly, these compounds had little effect on SDH, perhaps because a C2 amino acid is the preferred substrate of the enzyme. Meso-alanopine (2,2'-iminodi-

TABLE 7. Levels of some amino acids in muscle and Dharvnx o f A. aculeata

Muscle Pharvnx

Taurine 28.1 f 7.75 46.5 f 4.56

L-serine 15.4 * 1.76 4.1 * 0.69 L-threonine 9.5 f 1.04 6.3 f 0.16

Glycine 4.5 * 0.27 382.1 f 24.5 L-alanine 5.1 * 0.01 10.6 f 3.30 L-valine 0.7 f 0.09 1.3 f 0.42 Total amino acids 118.7 500.9

Results are expressed as prnol/g wet weight i SEM, n = 2. L- cysteine was not detected in the tissue samples.

pionic acid) and iminodiacetic acid were strong inhibitors, with low Kis, of pharynx SDH, whereas D-strombine, the normal product of the enzyme, showed very high K i s .

Product inhibition of the reverse reaction was observed at suboptimal (2 mM alanopine or 10 mM strombine) imino acid substrate levels. Muscle ADH activity was reduced by 63% in the presence of 20 mM L-alanine; 20 mM glycine decreased the rate of strombine oxidation by SDH by 50%. Pyruvate a t 10 mM was also inhibitory.

Tissue amino acid levels in A. aculeata The levels of some amino acids in muscle

and pharynx are shown in Table 7. Glycine was the major amino acid found in pharynx, a level of 382 pmolJg being 10.5-fold higher than Km(app) for glycine of SDH (Table 3). The levels of other amino acids in pharynx are too low to allow them to be considered physiological substrates for SDH. Overall amino acid levels were much lower in muscle largely because of a much reduced glycine

376 K.B. STOREY

pool. Ratios, [amino a~id]/K,~,~~), are 0.41, 0.32, 0.37, 0.01, and 0.01 for alanine, serine, threonine, valine, and glycine, respectively, suggesting that, in addition to alanine, serine and threonine might also act as physiological substrates for ADH.

DISCUSSION

Tissues of the sea mouse contained only a single cytoplasmic dehydrogenase acting at the pyruvate branchpoint, either the SDH enzyme found in pharynx and intestine or the ADH enzyme found in muscle, elytra, and nerve. No lactate dehydrogenase or octopine dehydrogenase activities were found. The role of SDH and ADH in vivo must therefore be to maintain cytoplasmic redox balance in all situations demanding glycolytic energy production. This could include roles in both anaerobic metabolism and burst muscular work. Alanopine/strombine production during anoxia has been documented in two bivalve species (Collicutt and Ho- chachka, '77; de Zwaan and Zurburg, '81); anoxic conditions requiring the activation of anaerobic energy generation could be en- countered by A. aculeata during burrowing in mud. Burst work would commonly be required of the muscles and pharynx of A. mu- leata during prey capture. Carnivorous polychaetes capture prey by means of an ev- ersible pharynx (or proboscis). The pharynx is rapidly protruded, and jaws at the end of the pharynx seize the prey. Protrusion of the pharynx is aided by a rapid increase in coe- lomic pressure brought about by the contrac- tion of body wall muscles. These rapid movementslcontractions of pharynx and body muscles required for capture of prey may likely be associated with glycolytic energy production utilizing ADH or SDH as the ter- minal enzyme of glycolysis.

The two imino acid dehydrogenases found in A. aculeata differ widely in their amino acid substrate specificities. Both, however, are well adapted for function in the amino acid environment of their own tissue. Phar- ynx SDH is highly specialized for the use of glycine, showing very high K,s and/or low Vmas for other amino acids. In addition, the very high intracellular levels of glycine in pharynx probably indicate that glycine is the only physiological substrate of the enzyme in vivo. Muscle ADH, in contrast, has a very high affinity for L-alanine and for several other C3 or C4 amino acids but a very low affinity for glycine. Alanine is likely to be

the major physiological substrate of the enzyme, as alanine levels are closely tied to glycolytic function, alanine being one of the major products of anaerobic metabolism in marine invertebrates. However, both serine and threonine could also be physiological substrates for the enzyme. Despite their dif- fering amino acid specificities, the keto acid specificities of the two enzymes were very similar with similar K,s for pyruvate and similar apparent K,s and Vmaxs for alternative keto acids. Such a similarity in keto acid specificity may result because the function of both enzymes is tied to the glycolytic production of pyruvate.

The substrate specificities and effects of inhibitors demonstrate differences in the substrate sites of pharynx SDH and muscle ADH. The amino acid substrate site of SDH is geared for the use of a Cz amino acid substrate, whereas ADH can utilize C3 or C4 amino acids but has low affinity for C2. The use of C3 amino acids by ADH probably ac- counts for the strong inhibition of ADH by D- or L-lactate which are structurally similar to L-alanine. In keto acid specificity, pharynx SDH and muscle ADH resemble the enzymes from other sources (Fields and Hochachka, '81; Dando et al., '81; Plaxton and Storey, '82a). The C2 glyoxylate was a poor substrate while the enzymes were capable of utilizing C4 and C5 keto acids but not Cg. Like ADH and SDH from other sources, the dicarbox- ylic acid, oxaloacetate, was a better alternative substrate for the enzymes than was the corresponding a-keto acid, a-ketobutyrate.

Substrate inhibitions of the enzymes in the forward direction were low in all cases. Like ADH from other sources (Plaxton and Storey, '82a) as well as brain type octopine dehydrogenase (Storey and Storey, '79) and H type lactate dehydrogenase (Long and Kaplan, '731, the enzymes from A. aculeata show substrate inhibition by pyruvate a t high levels. Alanine and glycine were also inhibitory at high concentrations, as has been described previously (Plaxton and Storey, '82a). The enzymes differed greatly in product inhibitions by imino acids. Muscle ADH was strongly inhibited by rneso-alanopine whereas pharynx SDH was only weakly inhibited by D-strombine. This low inhibition by product may be a feature designed to facilitate a high rate of glycolytic energy production during muscle work in pharynx, producing a rapid accumulation of strombine. Muscle-type octopine dehydrogenase and M-type lactate de-


hydrogenase are similarly only weakly inhibited by accumulated product (Storey and Storey, '79).

The amino acid substrate specificities of alanopine dehydrogenase and strombine dehydrogenase have now been studied for the enzymes from several marine invertebrate sources. These specificities appear to be highly variable. Sponge SDH utilized no amino acid other than glycine (Barrett and Butterworth, '81) whereas gastropod ADHs (Littorina littorea, Busycotypus canaliculatum) were highly specific for alanine (Plax- ton and Storey, '82a, b). The oyster adductor muscle enzyme, however, showed equivalent Kms and Vmaxs for both glycine and alanine (Fields and Hochachka, '81). Similar results were found for the adductor muscle enzyme from M. edulis (Dando, '81) as well as the muscle enzyme from Mercenaria mercenaria (Storey et al., '82). The alanopine dehydrogenase characterizing the soft tissues of M. edulis showed Kms of 10 and 50 mM for alanine and glycine, respectively, with high relative velocities for only alanine and cysteine. None of these enzymes closely resembles either the ADH or the SDH from the sea mouse. The variability in amino acid specificities amongst these enzymes may have several roots. First, despite the small number of species which have been studied, some phylogenetic differences are apparent. The ADHs of both gastropod species are highly specific for alanine, and the three bivalve adductor muscle enzymes show equivalent specificities for alanine and glycine. The substrate utilization patterns of A. aculeata SDH and ADH may therefore have a phylogenetic base, although this can only be tested by further studies on other species of polychaetes. Second, the enzyme specificity for amino acid may be evolutionarily altered in response to the makeup of the amino acid pool of different species or of different tissues within a species. Such may be the case for A. aculeata SDH, an enzyme with a very high glycine specificity being found in a tissue with extremely high glycine levels. The SDH in M. edulis adductor similarly occurs in a tissue where glycine is a dominant amino acid (Dando et al., '81). Third, enzyme specificity may be altered in response to the physiological role of the enzyme in vivo. For octopine dehydrogenase, enzyme utilization of L-arginine (compared to L-lysine or alternative guanidino amino acids) is greatly increased in species (ex. cephalopods, Pecten)

exhibiting a large argininetarginine phos- phate pool and a well-defined role for the enzyme in glycolytic energy production compared to those species such as sea anemones where enzyme role is less clear (Storey and Dando, '82). A similar reasoning may apply with respect to alanopinelstrombine dehydrogenases, substrate specificities becoming more narrow as the enzymes' physiological roles become more defined.

The characterization of an enzyme as an alanopine dehydrogenase versus a strombine dehydrogenase is beginning to become less clear as the enzymes from more species are studied. The sponge enzyme can clearly be called an SDH, and gastropod enzymes should probably be called ADHs although they do exhibit some glycine activity. Other enzymes, such as the bivalve adductor muscle enzymes, could clearly be called either ADH or SDH. In addition, as tissue-specific differences in enzyme compliment are uncov- ered, it is conceivable that we are perhaps not dealing with separate enzymes but rather with tissue-specific forms of a single enzyme (A. aculeata displaying two isozymic forms). This growing problem in nomenclature will have to be resolved; possibly enzyme names should be derived in accordance with the imino acid produced (alanopine or strombine) in vivo rather than according to in vitro exami- nations of enzyme substrate specificities.

ACKNOWLEDGMENTS

I wish to thank Dr. P. R. Dando, Marine Biological Association, Plymouth, and Dr. J. Tempe, C.N.R.A., Versailles, for helpful dis- cussions on the synthesis of meso-alanopine and D-strombine, and the staff of the M.B.A. for collection of A. aculeata. Amino acid determinations were performed by Mrs. C. Shay, Department of Biology, Carleton Uni- versity. Supported by an N.S.E.R.C. operat- ing grant and by N.R.C. contract OSU81- 00472.

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tissue‐specific alanopine dehydrogenase and strombine dehydrogenase from the sea mouse, aphrodite...

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