The effects of hydrostatic pressure on pertussis toxin-catalyzed ribosylation of G proteins from deep-living macrourid fishes.

To test the effects of hydrostatic pressure on the coupling of receptors to guanyl nucleotide binding reglatory proteins (G proteins) in transmembrane signaling, pertussis toxin (PTX)-catalyzed [32P]ADP-ribosylation was used to probe the guanyl nucleotide-binding proteins Gi and G(o) in brain membranes from four marine teleosts. These macrourids, Coryphaenoides pectoralis, Coryphaenoides cinereus, Coryphaenoides filifer and Coryphaenoides armatus, span depths from 200 to 5400 m. Pertussis toxin specifically labelled proteins of 39-41 kDa. The PTX-catalyzed [32P]ADP-ribosylation reaction was linear for 7 h. Added guanyl nucleotides (guanosine 5'-diphosphate (GDP) and guanosine 5'-O-(3-thiotriphosphate)(GTP[S])) at concentrations up to 1000 microM did not affect ribosylation at atmospheric pressure. Under basal conditions the Gi/G(o) protein population appears to be uncoupled from receptors and bound with GDP. Pressures up to 476 atm were tested in the absence and presence of added guanyl nucleotides, 100 microM GDP and 100 microM GTP[S]. [32P]ADP-ribosylation in brain membranes from the deeper-occurring C. cinereus, C. filifer and C. armatus was not inhibited by increased pressure in the presence of 100 microM GDP. Increasing pressure decreased ribosylation in brain membranes of C. pectoralis. In the presence of 100 microM GTP[S], increased pressure inhibited ribosylation in all species. Pressure appears to enhance the efficacy of GTP[S] in dissociating the heterotrimeric holoprotein.

Trimethylamine oxide stabilizes teleost and mammalian lactate dehydrogenases against inactivation by hydrostatic pressure and trypsinolysis.

Trimethylamine N-oxide (TMAO) is an organic osmolyte present at high levels in elasmobranchs, in which it counteracts the deleterious effects of urea on proteins, and is also accumulated by deep-living invertebrates and teleost fishes. To test the hypothesis that TMAO may compensate for the adverse effects of elevated pressure on protein structure in deep-sea species, we studied the efficacy of TMAO in preventing denaturation and enhanced proteolysis by hydrostatic pressure. TMAO was compared to a common 'compatible' osmolyte, glycine, using muscle-type lactate dehydrogenase (A(4)-LDH) homologs from three scorpaenid teleost fish species and from a mammal, the cow. Test conditions lasted 1 h and were: (1) no addition, (2) 250 mmol l(-)(1) TMAO and (3) 250 mmol l(-)(1) glycine, in the absence and presence of trypsin. Comparisons were made at 0. 1 and 101.3 MPa for the deeper occurring Sebastolobus altivelis, 0.1, 50.7 and 101.3 MPa for the moderate-depth congener S. alascanus, 0. 1 and 25.3 MPa for shallow-living Sebastes melanops and 0.1 and 50.7 MPa for Bos taurus. Susceptibility to denaturation was determined by the residual LDH activity. For all the species and pressures tested, 250 mmol l(-)(1) TMAO reduced trypsinolysis significantly. For all except S. altivelis, which was minimally affected by 101.3 MPa pressure, TMAO stabilized the LDH homologs and reduced pressure denaturation significantly. Glycine, in contrast, showed no ability to reduce pressure denaturation alone, and little or no ability to reduce the rate of proteolysis.

Evidence for heterotrimeric GTP-binding proteins in Toxoplasma gondii.

Toxoplasma gondii, an intracellular protozoan parasite, resides within a host-derived vacuole that is rapidly modified by a parasite-secreted membranous tubular network. In this study we investigated the involvement of heterotrimeric G proteins in the secretory pathway of T. gondii. Aluminum fluoride (AlFn), a specific activator of heterotrimeric G proteins, induced secretion from isolated tachyzoites of T. gondii in vitro, as seen by light optics and electron microscopy. In Western blot analyses, antibodies to G protein alpha subunits reacted with 39-42 kDa proteins from T. gondii isolates. Antibodies to G(o) alpha and Gs alpha coupled to the fluorescent probe fluorescein isothiocyanate localized to the paranuclear region of T. gondii. Gi3 alpha immunoprobes were confined to the cytoplasmic matrix of T. gondii and also labeled the parasitophorous vesicle. Fluorescein isothiocyanate-conjugated GA/1, an antipeptide antisera directed toward the GTP binding site common to G protein alpha subunits, was confined to the lateral cytoplasmic domain of the parasites where secretion is most prominent. In time-sequence studies using the GA/1 probe, the immunoreactive material shifted position during invasion of target cell to areas of active secretion.

Differential Susceptibility of Guanine Nucleotide-binding Proteins to Pertussis Toxin-catalyzed ADP-ribosylation in Brain Membranes of Two Congeneric Marine Fishes.

Pertussis toxin-catalyzed [32P]ADP-ribosylation was used to probe the guanine nucleotide binding regulatory proteins Gi and Go in brain membranes from two scorpaenid fishes, Sebastolobus alascanus and S. altivelis. The membranes of the two species exhibit a differential sensitivity to [32P]ADP-ribosylation produced by a fixed concentration of pertussis toxin. The membranes from the deeper-living S. altivelis consistently incorporated more [32P]ADP than the membranes from S. alascanus. Proteins of 39 and 41 kDa are specifically labeled in both species, corresponding to the apparent molecular masses of the α subunits of Gi and Go. At 5°C the ribosylation reaction is linear for at least 7 h. The pertussis toxin concentration-response relationship was evaluated with concentrations of pertussis toxin from 0 to 100 ng/ {mu}l. The extent of [32P]ADP-ribosylation was quantified by autoradiography and computer-assisted image analysis. The EC50 values for pertussis toxin were similar for the two species, but the maximum level of [32P]ADP-ribosylation was significantly greater in S. altivelis brain membranes. Because the heterotrimeric holoprotein is the substrate for ribosylation, the modulatory effects of the guanyl nucleotides GDP and GTPγS on the ribosylation were assessed. GDP increased [32P]ADP-ribosylation of the α subunits in S. altivelis. Only the highest concentration tested (1000 µM) increased [32P]ADP-ribosylation in S. alascanus brain membranes and only to a modest extent. Increasing concentrations of GTPγS suppressed [32P]ADP-ribosylation in S. alascanus brain membranes, presumably by promoting dissociation of the holotrimer. GTPγS had much less of an effect on the S. altivelis brain membranes. These differences in the extent of ADP-ribosylation and the modulatory effects of guanyl nucleotides may reflect different coupling efficiencies of G proteins and receptors. The expression of the α and ß subunits of Gi and Go in the two Sebastolobus species, the deep-sea morid teleost fish Antimora rostrata, and the rat were compared by Western immunoblotting of brain membranes with antipeptide antisera. Levels of Giα3 were 63% higher in brain membranes of S. altivelis than those in S. alascanus. The levels of Giα1, Giα2, Go and ß36 were similar in the two species. Although the complement of G proteins identified by the array of antisera used was similar in all the species, there appears to be additional diversity of α subunits in the teleost brain membranes. In fish, antiserum to Goα reacted with an additional 41 to 42 kDa protein that was not expressed in rat brain.

Proteolysis at pressure and HPLC peptide mapping of M4-lactate dehydrogenase homologs from marine fishes living at different depths.

1. The effects at 10 degrees C of moderate hydrostatic pressure (136 atm) on trypsinolysis of muscle-type (M4) lactate dehydrogenase homologs (LDH, EC 1.1.1.27, L-lactate:NAD+ oxidoreductase) from shallow- and deep-occurring marine fishes were examined by mapping the partial digests by reverse phase HPLC. 2. Comparison of peptide maps of digests generated at 1 and 136 atm revealed that increased pressure did not expose new cleavage sites in homologs of any of the species; no new peptides were generated. 3. Increased pressure did alter the relative amounts of peptides produced. The net effect of increased pressure was to increase the amount of peptides generated in the shallow-occurring species. For deep-living species pressure did not alter the net amount of peptides produced compared to the 15 min atmospheric pressure samples, although the relative amounts of some of the peptides changed. Incubation at 136 atm for 30 min decreased the net amount of peptides produced. 4. It is suggested that the effects of pressure on trypsinolysis may result from slight conformational changes in the substrate proteins.

A1 Adenosine Receptor Modulation of Adenylyl Cyclase of a Deep-living Teleost Fish, Antimora rostrata.

Low temperatures and high hydrostatic pressures are typical of the deep sea. The effects of these parameters on transmembrane signal transduction were determined through a study of the A1 adenosine receptor-inhibitory guanine nucleotide binding protein-adenylyl cyclase system in brain membranes of the bathyal teleost fish, Antimora rostrata (Moridae). The components of this system were analyzed at 5°C and 1 atm, and the role of the A1 receptor in the modulation of adenylyl cyclase was determined. The A1 selective radioligand N6-[3H]cyclohexyladenosine bound saturably, reversibly, and with high affinity. The Kd of N6-[3H]cyclohexyladenosine estimated from kinetic measurements was 1.11 nM; the Kd determined from equilibrium binding was 4.86 nM. [32P]ADP-ribosylation of brain membranes by pertussis toxin labeled substrates with apparent molecular masses of 39,000 to 41,000 Da. Basal adenylyl cyclase activity was inhibited in a concentration-dependent manner by the A1 adenosine receptor agonist N6-cyclopentyladenosine (IC50 = 5.08 µM). The inhibition of adenylyl cyclase activity was dependent upon GTP. Basal adenylyl cyclase activity was unaffected by 272 atm of pressure. The efficacy of 100 µM N6-cyclopentyladenosine as an inhibitor of adenylyl cyclase was the same at atmospheric pressure and at 272 atm. The inhibition of adenylyl cyclase by the agonist 5'-N-ethylcarboxamidoadenosine (100 µM) at 272 atm was twice that observed at atmospheric pressure. Although consideration of the effects of low temperature and high hydrostatic pressure on acyl chain order suggest that deep-sea conditions will perturb membrane function, signal transduction by the A1 receptor system of the bathyal fish A. rostrata is not disrupted by deep-sea conditions.

Inactivation of NAD-dependent dehydrogenases from shallow- and deep-living fishes by hydrostatic pressure and proteolysis.

Cytoplasmic malate dehydrogenase [L)-malate:NAD+ oxidoreductase, EC 1.1.1.37) and glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase, EC 1.2.1.12) homologues from two shallow-living and three deep-living fishes were examined for the effects of hydrostatic pressure on enzyme activity and susceptibility to inactivation by proteinases. These studies were done to determine whether malate dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase homologues show similar patterns of adaptation to hydrostatic pressure as seen in lactate dehydrogenase (L-lactate:NAD+ oxidoreductase, EC 1.1.1.27) homologues from the same species (Hennessey, J.P., Jr. and Siebenaller, J.F. (1987) J. Exp. Zool. 241, 9-15). Fish malate dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase homologues are much less susceptible to inactivation by hydrostatic pressure than are lactate dehydrogenase homologues from the same species. This difference in susceptibility to inactivation by hydrostatic pressure may be due to the decreased number of intersubunit contacts in malate dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase homologues relative to lactate dehydrogenase homologues. Inactivation of malate dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase homologues by proteinases, both at atmospheric pressure and at elevated hydrostatic pressure, is less than for lactate dehydrogenase homologues from the same species. This suggests that the structural characteristics and conformational perturbations that are responsible for the susceptibility of lactate dehydrogenase to proteolytic inactivation are not found in malate dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase homologues of the same species.

Coenzyme binding ability of homologs of M4-lactate dehydrogenase in temperature adaptation.

Temperature effects on dissociation constants (Kd), binding enthalpies and apparent Michaelis constants (Km) for NADH, plus Arrhenius activation energies (Ea), substrate turnover numbers (kcat), and NADH 'on' constants (k1) were measured or calculated for M4-lactate dehydrogenase homologs from deep-sea, midwater, shallow-water temperate, and shallow-water tropical teleost fishes, and a mammal. At any single measurement temperature, Km and kcat values were significantly higher for groups adapted to lower temperatures. This pattern of Km values and temperature illustrates a strong evolutionary conservation of Km of NADH. When determined at the average body temperature of each species, the Km values are very similar, resulting in the preservation of the catalytic capacity and regulatory properties of these enzyme homologs at their in situ temperatures. In contrast, Kd values, while varying considerably among species, are not significantly different among the different groups at any one temperature. The ratio of Km to Kd tends to follow a phylogenetic pattern rather than a pattern of environmental adaptation. Thus, evolutionary adjustments in Km are not directly the result of changes in cofactor binding. All the rate constants involved in determining the Km and Kd of NADH (kcat, k1 and k-1) can be modified.

Comparison of the binding properties of A1 adenosine receptors in brain membranes of two congeneric marine fishes living at different depths.

The binding properties of A1 adenosine receptors in brain membranes were compared in two congeneric marine teleost fishes which differ in their depths of distribution. Adenosine receptors were labeled using the A1 selective radioligand [3H]cyclohexyladenosine ([3H]CHA). The A1 receptor agonist [3H]CHA bound saturably, reversibly and with high affinity to brain membranes prepared from Sebastolobus altivelis and S. alascanus; however, the mean Kd values differed significantly (Figs. 1-3, Table 1). Saturation data fit to a one site model indicated that the A1 receptor in S. alascanus exhibited a higher affinity (Kd = 1.49 nM) for [3H]CHA whereas A1 receptors in S. altivelis exhibited a significantly lower affinity (Kd = 3.1 nM). Moreover, S. altivelis, but not S. alascanus, parameter estimates for [3H]CHA binding to two sites of receptor were obtained (Fig. 3, Table 1). The mean dissociation constant values for the high and low affinity sites for [3H]CHA in S. altivelis were 0.43 nM and 16.3 nM, respectively. In equilibrium competition experiments the adenosine analogs R-phenylisopropyladenosine (R-PIA), N-ethylcarboxamidoadenosine (NECA) and S-phenylisopropyladenosine (S-PIA) all displayed higher affinities for A1 receptors in S. alascanus as compared to S. altivelis brain membranes (Table 2, Fig. 6). The specific binding of [3H]CHA was significantly increased by 0.1 and 1.0 mM MgCl2 in both fishes; however, the sensitivity (95-131% increase) of S. altivelis to this effect was significantly greater than that of S. alascanus (48-91% increase) (Fig. 5). The results of kinetic, equilibrium saturation and equilibrium competition experiments all suggest that A1 adenosine receptors of S. altivelis and S. alascanus brain membranes differ with respect to their affinities for selected adenosine agonists.

Pressure-adaptive differences in proteolytic inactivation of M4-lactate dehydrogenase homologues from marine fishes.

The inactivation by hydrostatic pressure of muscle-type lactate dehydrogenase (M4-LDH, EC 1.1.1.27; L-lactate: NAD+ oxidoreductase) homologues from five shallow-living and six deep-living marine teleost fishes was compared. The pressures which inactivate these enzymes are much higher than the pressures experienced by any of the species. To determine whether hydrostatic pressure effects on protein aggregation state and conformation might influence proteolysis, the inactivation of LDH by the proteases, trypsin (EC 3.4.21.4) and subtilisin (EC 3.4.4.16) was determined at atmospheric pressure and 1,000 atm pressure. At 10 degrees C and atmospheric pressure, the enzymes of the shallow-living fishes are inactivated four times faster by trypsin and three times faster by subtilisin than are the homologues of the deep-living species. At 1,000 atm pressure, the homologues of shallow-occurring fishes were inactivated 28 to 64% more than predicted from the summed effects of denaturation by 1,000 atm pressure and tryptic inactivation at atmospheric pressure. In contrast, the homologues of the deep-sea species were inactivated by trypsin 0 to 21% more than expected. At 1,000 atm, inactivation by subtilisin increased to a similar degree for enzymes from both deep- and shallow-living species. However, at 1,000 atm, the M4-LDH homologues of the deep-sea species lost less activity (55.3%) than did the homologues of the shallow species (86.4%). In comparisons made at 200 atm, a pressure typical of the habitat of the deep-occurring species, tryptic inactivation of the LDH of the shallow-living Sebastes melanops was increased 14%. No pressure inactivation of the enzyme is evident at 200 atm.(ABSTRACT TRUNCATED AT 250 WORDS)

Phylogenetic distribution of [3H]cyclohexyladenosine binding sites in nervous tissue.

The specific binding of the A1 adenosine receptor ligand, [3H]CHA, was investigated in membrane fractions prepared from brains of eleven vertebrate species and ganglia of four invertebrate species. Substantial amounts of specific [3H]CHA binding sites were demonstrated in brain membranes of all vertebrate species examined; however, [3H]CHA binding sites were not detectable in nervous tissue of the invertebrate species studied. The densities of [3H]CHA binding sites in vertebrate brains increase in higher vertebrates. Moreover, the pharmacological characteristics of the site labeled by [3H]CHA in two divergent classes of vertebrates were similar. The broad phylogenetic distribution of A1 adenosine receptors in primitive as well as advanced vertebrate species suggests a fundamental role for adenosine in neuronal modulation.

Characterization of muscarinic cholinergic receptors in the crab nervous system.

The selective muscarinic antagonist L-[3H]-quinuclidinyl benzilate (L-[3H]QNB) binds reversibly and with high affinity (KD = 0.3 nM) to a single population (Bmax = 105 fmol/mg protein) of specific sites in nervous tissue of the crab Cancer magister. The binding site is stereoselective; (-)QNB is over 200 times more potent than (+)QNB as an inhibitor of specific L-[3H]QNB binding. The muscarinic antagonists scopolamine and atropine are over 10,000 times more potent inhibitors of L-[3H]QNB binding than the nicotinic antagonists decamethonium and d-tubocurarine. The muscarinic agonists oxotremorine, pilocarpine, arecoline, and carbachol also compete effectively for the L-[3H]QNB binding site. This pharmacological profile strongly suggests the presence of classical muscarinic receptors in the crab nervous system. These receptors are localized to nervous tissue containing cell bodies and neuropil, whereas specific L-[3H]QNB binding is low or absent in peripheral nerve, skeletal muscle, and artery.

Stereoselective L-[3H]quinuclidinyl benzilate-binding sites in nervous tissue of Aplysia californica: evidence for muscarinic receptors.

The muscarinic antagonist L-[3H]quinuclidinyl benzilate (L-[3H]QNB) binds with a high affinity (Kd = 0.77 nM) to a single population of specific sites (Bmax = 47 fmol/mg of protein) in nervous tissue of the gastropod mollusc, Aplysia. The specific L-[3H]QNB binding is displaced stereoselectively by the enantiomers of benzetimide, dexetimide, and levetimide. The pharmacologically active enantiomer, dexetimide, is more potent than levetimide as an inhibitor of L-[3H]QNB binding. Moreover, the muscarinic cholinergic ligands, scopolamine, atropine, oxotremorine, and pilocarpine are effective inhibitors of the specific L-[3H]QNB binding, whereas nicotinic receptor antagonists, decamethonium and d-tubocurarine, are considerably less effective. These pharmacological characteristics of the L-[3H]QNB-binding site provide evidence for classical muscarinic receptors in Aplysia nervous tissue. The physiological relevance of the dexetimide-displaceable L-[3H]QNB-binding site was supported by the demonstration of the sensitivity of the specific binding to thermal denaturation. Specific binding of L-[3H]QNB was also detected in nervous tissue of another marine gastropod, Pleurobranchaea californica. The characteristics of the Aplysia L-[3H]QNB-binding site are in accordance with studies of numerous vertebrate and invertebrate tissues indicating that the muscarinic cholinergic receptor site has been highly conserved through evolution.

Pressure inactivation of tetrameric lactate dehydrogenase homologues of confamilial deep-living fishes.

The susceptibility to inactivation by hydrostatic pressure of the tetrameric muscle-type (M4) lactate dehydrogenase homologues (LDH, EC 1.1.1.27; L-lactate: NAD+ oxidoreductase) from six confamilial macrourid fishes was compared at 4 degrees C. These marine teleost fishes occur over depths of 260 to 4815 m. The pressures necessary to half-inactivate the LDH homologues are related to the pressures which the enzymes are exposed to in vivo; higher hydrostatic pressures are required to inactivate the LDH homologues of the deeper-occurring macrourids. The resistance of the LDH homologues to inactivation by pressure is affected by protein concentration. After an hour of incubation at pressure, the percent remaining activity approaches an asymptomatic value. The inactivation of the macrourid LDH homologues by pressure was not fully reversible. Assuming that inactivation by pressure was due to dissociation of the native tetramer to monomers, apparent equilibrium constants (Keq) were calculated. Volume changes (delta V) were calculated over the range of pressures for which plots ln Keq versus pressure were linear. The delta V of dissociation values of the macrourid homologues range from -219 to -439 ml mol-1. Although the hydrostatic pressures required to inactivate the LDH homologues of the macrourid fishes are greater than those which the enzymes are exposed to in vivo, the pressure-stability of these enzymes may reflect the resistance of these enzymes to pressure-enhanced proteolysis in vivo.

Structural comparison of lactate dehydrogenase homologs differing in sensitivity to hydrostatic pressure.

The muscle-type (M4) lactate dehydrogenases (L-lactate: NAD+ oxidoreductase EC 1.1.1.27) of two teleost fishes, Sebastolobus alascanus and Sebastolobus altivelis , differ in the susceptibility of ligand binding to perturbation by moderate hydrostatic pressures. The enzyme homologs were purified by affinity chromatography. The amino-acid compositions of these enzymes are virtually identical. The proteins were digested with trypsin and the peptide mixtures mapped using reverse-phase HPLC. Although there was variation in elution times of some peaks, the amino-acid compositions of the fractions from the two profiles were highly similar. Only one clear difference in amino-acid composition was found and this peptide was sequenced using the manual dansyl-Edman method. The enzyme of S. alascanus , which is susceptible to pressure-perturbation, had a histidine at position 115; the S. altivelis enzyme had an asparagine. Ionization of histidine is affected by pressure and may be involved in the differences between the two lactate dehydrogenase homologs. There is no covalently bound phosphate associated with either enzyme, and thus phosphorylation cannot account for the differences between the enzyme homologs. Acquisition of pressure-tolerance appears to involve only minor changes in primary structure.

Comparison of the D-lactate stereospecific dehydrogenase of Limulus polyphemus with active-site regions of L-lactate dehydrogenases.

Lactate dehydrogenase (D-lactate:NAD+ oxidoreductase, EC 1.1.1.28) from the horseshoe crab, Limulus polyphemus, a dimeric enzyme stereospecific for D-lactate, has been purified by affinity chromatography. Maleyl tryptic peptides containing arginine residues isolated from the Limulus enzyme have been characterized and sequenced. The small peptides obtained from similarly treated L-lactate-specific enzyme homologs define major portions of the substrate and coenzyme binding regions and are virtually identical among L-lactate-specific enzymes. Although the six small peptides and free arginine isolated from the Limulus enzyme indicate that the small number of arginine tryptic peptides are located in a few discrete consecutive clusters similarly to the L-lactate dehydrogenases, the peptides nevertheless show no obvious sequence homology to the corresponding peptides from L-lactate dehydrogenases. These results indicate that this lactate dehydrogenase of altered substrate specificity either evolved with major rearrangements of the active site if it evolved from an L-lactate dehydrogenase, or that D-lactate dehydrogenases have evolved from a different protein. The results contradict proposed models which suggest that minor changes in the spatial orientation of pyruvate resulting from minimal rearrangement of the active site could accommodate the change in substrate specificity.

Biochemical studies of aminopeptidase polymorphism in Mytilus edulis. II. Dependence of reaction rate on physical factors and enzyme concentration.

Enzymatic parameters of aminopeptidase-I that may be sensitive to temperature an solute variations were investigated to provide a functional explanation for specific activity differences among genotypes in natural populations. The effect of temperature on the apparent Km of L-leucyl-4-methoxy 2-naphthylamide and th dipeptide phenylalanyl-glycine was small, especially between 10 and 25 C. The apparent Km varied only between 36.7 and 49.8 microM at these temperatures and the six common genotypes did not differ in temperature-dependent substrate affinities. While pH had a significant effect on Km, no differences among genotypes were observed. Activation enthalpies were also identical among genotypes. Thermal inactivation was slowest at 15 C and the same for all genotypes. Of 18 tested amino acids, only phenylalanine inhibited aminopeptidase-I; K1 values ranged from 1.2 to 0.8 mM and were the same for all genotypes. Small differences among genotypes were detected in the inhibitory effect of zinc. The concentration of aminopeptidase-I enzyme was the same for all genotypes in a population exposed to oceanic salinity, but the concentration of Lap 94/94 was 15% lower than that of other genotypes in a population experiencing estuarine salinity. Genotypes with the Lap 94 allele exhibited higher apparent Kcat values in all population samples.The probable genotype-dependent effects of enzyme concentration and Kcat differences are discussed with regard to maintenance of the polymorphism and genetic differences among populations.