Busycotypus Canaliculatum Research Articles

Organ-specific regulation of phosphofructokinase during facultative anaerobiosis in the marine whelk Busycotypus canaliculatum

The kinetic properties of 6-phosphofructo-1-kinase (PFK) were assessed in five organs (ventricle, radular retractor muscle, gill, hepatopancreas, and kidney) of aerobic and anoxic (21 h in N2-bubbled seawater) whelks, Busycotypus canaliculatum. The enzyme in all organs showed a stable modification of kinetic parameters as a result of exposure of the animal to anoxic conditions. In most cases these changes were consistent with the conversion of the enzyme to a less active form in the anoxic organ. In ventricle, for example, the anoxic enzyme form showed significant changes, including a 36% increase in the value of the substrate affinity constant (S0.5) for Mg∙ATP, a 19% increase in S0.5 fructose-6-phosphate, a 57% increase in the 50% inhibition value (I50) for phosphoenolpyruvate, a 30% increase in I50 citrate, and a fivefold increase in the activator constant (Ka) for fructose-2, 6-bisphosphate, as compared with the aerobic enzyme. Analysis of the time course of anoxia-induced modification of PFK showed that changes to the properties of gill PFK were accomplished within 2 h of the exposure to N2-bubbled seawater, whereas changes to ventricle PFK required up to 8 h. In vitro incubation of ventricle homogenates with Mg∙ATP plus protein kinase second messengers or with Mg2+ plus added protein phosphatases showed that the aerobic enzyme form was modified by protein kinase action with an increase in Ka fructose-2,6-bisphosphate that mimicked the effect of the aerobic to anoxic transition on the enzyme. Phosphatase action on the anoxic enzyme form had the opposite effect. The data suggest that the modification of PFK properties under anoxia in whelk organs is due to protein phosphorylation of the enzyme. Such covalent modification of PFK and other enzymes, notably pyruvate kinase, coordinates the anoxia-induced glycolytic rate depression and overall metabolic arrest that is a prominent feature of facultative anaerobiosis in marine molluscs.

Anaerobiosis and organ-specific regulation of glycolysis in a marine whelk

Changes in the levels of glycolytic intermediates, adenylates, phosphagen, and substrates and products of anaerobic metabolism were monitored over the initial hours of anaerobiosis in radular retractor muscle, foot muscle, and gill of the marine whelk Busycotypus canaliculatum. In all organs, changes in glycolytic intermediates upon exposure to N2-bubbled seawater indicated a biphasic response by glycolytic rate to anoxia: an immediate activation (within 30 min to 1 h) followed after 1–4 h by glycolytic rate depression. In both cases, regulatory control at phosphofructokinase and pyruvate kinase was indicated. The accumulation of anaerobic end products differed between the three organs. In radular retractor and gill, the ratio alanine succinate produced was 2:1 (but with higher amounts of both products in radular retractor), whereas in foot the ratio was 12.5:1. Foot muscle showed a large depletion of arginine phosphate reserves early in anoxia but little perturbation of adenylate pools, whereas gill and radular retractor showed a much lower net use of phosphagen but substantial depletion of organ ATP content. The data support several conclusions: (i) metabolic rate depression in the whelk is initiated within 1–2 h of anoxia exposure, (ii) mechanisms of metabolic depression override allosteric activating effects by phosphagen and adenylates on phosphofructokinase, and (iii) inhibition of flux through pyruvate kinase parallels the course of metabolic depression, not changes in carbon partitioning at the phosphoenolpyruvate branch point.

Influence of pH on the regulatory properties of aerobic and anoxic forms of pyruvate kinase in a marine whelk

AbstractThe effects of pH on the kinetic properties of pyruvate kinase were determined for both the aerobic and anoxic (16 hours exposure to N2‐bubbled seawater) enzyme forms from foot muscle and gill of the marine whelk, Busycotypus canaliculatum. The two enzyme forms from each tissue had distinctly different properties; compared to the aerobic enzyme, the anoxic enzyme form had significantly increased values for S0.5 phosphoenolpyruvate and Ka fructose‐1,6‐bisphosphate and greatly reduced I50 values for L‐alanine. Effects of pH on kinetic constants were assessed at pH 6.6, 7.0, and 7.5. Both the aerobic and anoxic forms of pyruvate kinase in whelk foot showed minimal values for S0.5 PEP and Ka FBP at pH 7.0; these rose by up to twofold at the higher or lower pH values. I50 values for alanine and Mg.ATP were strongly affected by low pH; I50 Mg.ATP fell by tenfold at pH 6.6 compared to pH 7.0, whereas I50 ALA dropped eightfold for the aerobic and 2.5‐fold for the anoxic enzyme form over the same pH change. The qualitative effects of pH change were similar for the anoxic enzyme form from gill. PK‐aerobic from gill behaved differently, however; as pH decreased, S0.5 PEP increased, whereas Ka FBP and I50 ALA both decreased. Furthermore, inhibition by Mg‐ATP occurred only at low pH. Although anoxia‐induced phosphorylation of PK is clearly the primary mechanism for changing the activity state of PK during anaerobiosis, the results suggest that the progressive fall in intracellular pH that occurs during anoxia would enhance the inactivation of PK activity in whelk organs. In particular, the effect of low pH in increasing the sensitivity of the anoxic enzyme forms to inhibitors could facilitate the anoxia‐induced changes in carbon flow at the phosphoenolpyruvate branchpoint.

Organ-Specific Analysis of the Time Course of Covalent Modification of Pyruvate Kinase during Anaerobiosis in a Marine Whelk

The progress of anoxia-inducedphosphorylation of pyruvate kinase (PK) was monitored in four organs of the whelk Busycotypus canaliculatum over the course of 21 h of exposure to N2-bubbled seawater. In all cases, PK modification resulted in a decrease in affinity for phosphoenolpyruvate (PEP) (the substrate affinity constant, Sos, rose by 3–6-fold) and a strong increase in inhibition by alanine (the inhibitor constant, I50, decreased to 0.1%–13% of the aerobic value). However, the time course of kinetic changes was distinctly different for each of the three isozymic forms tested (ventricle, foot, and hepatopancreas/kidney), and, in all cases, anoxia-induced modification of the I50(Ala) occurred much more rapidly than did changes in S0.5(PEP). Enzyme modification occurred most rapidly in hepatopancreas and kidney with half-times for the decrease in I50(Ala) of 20–25 min and for the increase in S0.5(PEP) of 100 min. Comparable valuesfor ventricle were 110 and 210 min and for foot were 225 and 550 min for I50(Ala) and S.5(PEP), respectively. With two phosphorylation sitesper subunit, these data suggest that anoxia-induced enzyme phosphorylation occurs first at a site that influences I50(Ala). The initial response to anoxia exposure by PK in both ventricle and foot included a change in kinetic properties that was opposite to the final result after long-term anoxia. In ventricle, for example, S0.5(PEP) decreased and I50(Ala) increased within the first 30 min; by 2 h, however, these changes had reversed and kinetic parameters were progressing toward the anoxic endpoints. These data are consistent with a biphasic response by metabolism over the aerobic-anaerobic transition in ventricle and foot: an initial activation of PK (and ofglycolytic rate) as conditions become increasingly hypoxic, followed by PK inactivation (and glycolytic rate depression) to permit longterm anaerobiosis.

Mechanisms of glycolytic control during facultative anaerobiosis in a marine mollusc: tissue-specific analysis of glycogen phosphorylase and fructose-2,6-bisphosphate

Changes in the activity of glycogen phosphorylase and the content of fructose-2,6-bisphosphate (F-2, 6-P2) were monitored in tissues of the whelk, Busycotypus canaliculatum, over a 21-h course of environmental anoxia. Tissue-specific responses to anoxia were seen with respect to phosphorylase content: in the radular retractor muscle and foot, the content of phosphorylase a expressed rose rapidly over the initial hours of anoxia (maximal increases were 4.3- and 2.5-fold, respectively) while in the gill, content dropped 2-fold during anoxia. Phosphorylase content was modulated by two mechanisms, changes in the percentage of enzyme in the active a form and changes in the total amount (a + b) of enzyme expressed. Anoxia stimulated a dramatic reduction in F-2,6-P2 content in five tissues. In the ventricle, content fell by 224-fold with a t1/2 of only 35 min. Levels in gill, radular retractor, hepatopancreas, and kidney fell to 2.5–3.5% of control values within the first 8 h of anoxia. F-2,6-P2 content in foot muscle was not altered during anoxia. Changes in glycogen phosphorylase activities and F-2,6-P2 contents help to produce tissue-specific responses of glycolysis to environmental anoxia that acknowledge competing metabolic demands including metabolic rate depression, changes in fuel use, anaerobic energy needs, and carbohydrate use for anabolic purposes.

Aspartate activation of pyruvate kinase in anoxia tolerant molluscs

1. 1. The effects of the amino acid, aspartate, as an allosteric modifier of pyruvate kinase (PK) were surveyed in tissue extracts from 18 species of animals. 2. 2. Aspartate was a strong activator of PK in selected tissues of gastropod and bivalves molluscs, particularly amongst anoxia-tolerant species; with one exception, a polychaete worm, aspartate had no effect on PK from non-molluscan species tested including marine, freshwater and terrestrial forms. 3. 3. Amongst gastropods, aspartate activation of PK was confined to selected soft tissues, most often the hepatopancreas; in the whelk, Busycotypus canaliculatum, hepatopancreas, gill and kidney PK all showed the effect. 4. 4. Amongst bivalves, aspartate activated PK in all tissues tested of the oyster, Crassostrea virginica, but activated only hepatopancreas PK in Mytilus edulis and foot and phasic adductor muscle PK in Mercenaria mercenaria. 5. 5. Kinetic properties of C. virginica adductor and M. mercenaria foot muscle PK were further examined; for C. virginica adductor PK aspartate (5 mM) raised enzyme maximal velocity by 2.8-fold and lowered S 0.5 for P-enolpyruvate by 36%. 6. 6. Aspartate ( K a = 4.14 ± 1.15 mM) and fructose-1,6-P 2 ( K a = 0.075 ± 0.005 mM) showed strong synergistic effects in their activations of C. virginica adductor PK. 7. 7. Proposed functions for the unique activation of PK by aspartate in anoxia tolerant molluscs are (a) in gluconeogenic tissues (hepatopancreas), allosteric activators (aspartate, fructose-1,6-P 2) are key to regulation of the aerobic (dephosphorylated) form of PK with respect to glycolytic vs gluconeogenic carbon flux; and (b) aspartate activation of PK provides co-ordinated control of the coupled anaerobic fermentations of aspartate → succinate and glycogen → alanine.

Glycolytic enzyme binding and metabolic control in anaerobiosis

Metabolic rate depression is a key survival strategy used by facultative anaerobes for enduring periods of environmental anoxia. In determining the molecular mechanisms of this phenomenon the role of enzyme binding to the subcellular particulate fraction was assessed in muscle tissues (ventricle and foot) of the anoxia tolerant marine gastropod,Busycotypus canaliculatum. Using two different methodologies for preparation, soluble versus particulate fractions of muscle were separated and assayed for their contents of eight glycolytic enzymes. Preparations from anoxic animals showed decreased percentages of enzymes associated with the particulate fraction as compared to controls; this was particularly pronounced for hexokinase and aldolase. A return to aerated seawater reversed this effect, and increased enzyme binding to the particulate fraction. The absence of a Pasteur effect in animal facultative anaerobes may be due, in part, to an anoxia-induced dissociation of enzymes from the particulate fraction of the cell promoting a decrease in glycolytic rate.

Purification and properties of aerobic and anoxic forms of pyruvate kinase from the hepatopancreas of the channelled whelk, Busycotypus canaliculatum

Aerobic and anoxic variants of pyruvate kinase (termed PK-aer and PK-anx) from the hepatopancreas of the gastropod mollusc, Busycotypus canaliculatum, were purified to apparent homogeneity with final specific activities of 14 and 2.3 units/mg protein, respectively. Both enzymes were homotetramers of the same molecular weight. The enzymes also showed equivalent affinities for ADP (0.22 m m) and very similar affinities for Mg 2+, Mn 2+, K +, and NH 4 +. PK-aer and PK-anx differed strongly, however, in maximal enzyme velocity ( V max 9-fold higher for PK-aer), in affinity for P-enolpyruvate (PEP 0.5 = 0.38 m m for PK-aer and 1.1 m m for PK-anx), and in the effects of activators and inhibitors on the enzymes. PK-aer was much more strongly stimulated by fructose-1,6-P 2 and aspartate as activators (a 19- and 32-fold activation of enzyme velocity at sub-saturating PEP levels versus only 4.1- and 2.6-fold activation for PK-anx, respectively). K a for fructose-1,6-P 2 was 3-fold lower (0.16 μ m) for PK-aer than for PK-anx (0.48 μ m), but K a for aspartate was the same for both enzymes (1.5 m m). Activators decreased the PEP 0.5 (to 0.05 m m for PK-aer and 0.07 m m for PK-anx), relieved inhibitions by alanine, Mg ATP, ADP, and P i, and, when added together, showed a strong synergistic activation of PK-aer (but not PK-anx). The kinetic differences between PK-aer and PK-anx are similar to those of the dephosphorylated versus phosphorylated forms of PK from other sources, including those of red muscle PK of B. canaliculatum, and indicate that the change in enzyme form brought about during anaerobiosis may be due to enzyme phosphorylation. The powerful activation of hepatopancreas PK by aspartate is a novel regulatory control of the enzyme. Aspartate is one of the substrates of anaerobic energy production in marine molluscs and its effects on the enzyme may be important in a tissue where inactivation of PK can occur for one of two reasons: anaerobiosis or gluconeogenesis.

Tissue specific isozymes of pyruvate kinase in the channelled whelkBusycotypus canaliculatum: enzyme modification in response to environmental anoxia

1. Isoelectrofocusing and cellulose acetate electrophoresis demonstrated the presence of three tissue specific isozymic forms of pyruvate kinase (PK) occurring in red muscle (ventricle, radula retractor), white muscle (foot, mantle) and soft tissues (gill, kidney, hepatopancreas) of the channelled whelk,Busycotypus canaliculatum. 2. Under the assay conditions used the three isozymes differed kinetically with respect to fructose-1,6-bisphosphate activation: red muscle PK was insensitive, white muscle PK was slightly activated and soft tissue PK was strongly activated. 3. Kinetic and physical properties of the enzyme in all tissues were modified when PK was isolated from whelks exposed to 21 h of anoxia stress. Compared to PK from aerobic tissues, the enzymes from anoxic animals showed a decrease in pI of 0.4 pH unit and an increase in anodal migration on cellulose acetate. Anoxia reduced the maximal activity of PK in all tissues and increased the activity ratios at high versus low phosphoenolpyruvate (PEP) concentrations. White muscle and soft tissue PK from anoxic whelks also showed increased activation by fructose-1,6-biphosphate compared to aerobic animals. 4. Maximal activities and activity ratios at high versus low PEP for phosphoenolpyruvate carboxykinase (PEPCK) were not altered as a result of anoxia stress. 5. The anoxia-induced in vivo modification of PK appears to be the result of enzyme phosphorylation during anoxia and this covalent modification is probably a major factor limiting PK activity during anoxia and promoting the carboxylation of PEP via PEPCK.

Phosphofructokinase from foot muscle of the whelk, Busycotypus canaliculatum: Evidence for covalent modification of the enzyme during anaerobiosis

Phosphofructokinase (PFK) was purified from foot muscle of aerobic and anaerobic (24 h of anoxia) whelks, Busycotypus canaliculatum. Fructose-6-P kinetics were sigmoidal at pH 7.0 with affinity constants, S 0.5, of 2.18 ± 0.10 ( n H = 2.5 ± 0.1) and 2.48 ± 0.13 m m ( n H = 2.7 ± 0.1) for the enzyme from aerobic versus anaerobic muscle. Affinity for ATP, like that for fructose-6-P, did not differ for the two enzymes (0.031 ± 0.003 for the aerobic vs 0.041 ± 0.007 m m for the anaerobic enzyme), but S 0.5 for Mg 2+ was significantly different for the two enzymes (0.060 ± 0.006 vs 0.130 ± 0.020 m m). Whelk muscle PFK was activated by NH 4 +, P i, AMP, ADP, and fructose-2,6-P 2. NH 4 + and fructose-2,6-P 2 were less effective activators of PFK from anoxic muscle, with apparent K a 's 1.6- and 3.5-fold higher for the anaerobic vs aerobic enzyme. Activators decreased S 0.5 for fructose-6-P and reduced n H. With the exception of fructose-2,6-P 2, the effects of activators on S 0.5 were the same for the enzyme from aerobic and anaerobic muscle; fructose-2,6-P 2 at 2.5 μ m reduced S 0.5 by only 3.3-fold for the anaerobic enzyme compared to 5.5-fold for the aerobic enzyme. ATP was a strong substrate inhibitor of PFK; the enzyme from anaerobic muscle showed greater ATP inhibition, with I 50's 1.5- to 2.0-fold lower than those for the aerobic enzyme. The kinetic differences between PFK from anaerobic versus aerobic foot muscle (stronger ATP inhibition and decreased sensitivity to activators for the anaerobic enzyme) were consistent with kinetic differences reported for the phosphorylated versus dephosphorylated forms, respectively, of PFK in other systems. Treatment of PFK from anaerobic muscle with alkaline phosphatase resulted in a decrease in the K a for fructose-2,6-P 2 to a level similar to that of the aerobic enzyme. The physiological stress of anoxia may, therefore, induce a covalent modification of PFK.

Purification and properties of aerobic and anoxic forms of pyruvate kinase from red muscle tissue of the channelled whelk, Busycotypus canaliculatum.

Aerobic and anoxic variants of radular retractor muscle pyruvate kinase (PK-aerobic and Pk-anoxic) from the gastropod mollusc, Busycotypus canaliculatum, were purified to homogeneity and respective specific activities of 368 and 186 mumol of product min-1 mg protein-1. Both PK variants were apparent homotetramers with native molecular masses of about 235 kDa, but differed in several other physical characteristics including pI (5.81 +/- 0.06 for PK-aerobic, 5.42 +/- 0.03 for PK-anoxic) and chromatographic behavior on several columns used during their respective purifications. The two enzymes differed greatly in several kinetic properties. Affinity for phosphoenolpyruvate was more than tenfold greater for PK-aerobic (K0.5 = 0.067 +/- 0.002 mM; h = 0.99 +/- 0.10), whereas the cooperative effect for phosphoenolpyruvate binding was greatly enhanced for PK-anoxic (K0.5 = 0.85 +/- 0.02 mM, h = 2.57 +/- 0.01). Although the affinities for the second substrate, ADP, were identical for both enzyme forms (apparent Km = 0.25 mM) pK-anoxic showed greater substrate inhibition by high concentrations of ADP. Likewise, affinities for K+ and Mg2+ were similar but PK-anoxic showed a greater degree of cooperativity with Mg2+ (h = 2.50 +/- 0.02) than did PK-aerobic (h = 1.70 +/- 0.06). Saturating concentrations of fructose 1,6-bisphosphate (50 microM) activated PK-anoxic resulting in an enzyme with properties similar to fructose-1,6-bisphosphate-activated PK-aerobic, with K0.5 values for phosphoenolpyruvate of about 0.04 mM and Hill coefficients of 1.1. PK-anoxic showed much stronger regulation by the allosteric inhibitors MgATP, phenylalanine, proline and alanine. Fructose 1,6-bisphosphate partially relieved the inhibitions by ADP, MgATP, alanine, proline and arginine phosphate of both enzyme forms. However, at 0.1 mM phosphoenolpyruvate PK-aerobic was much more sensitive to activation by fructose 1,6-bisphosphate, Ka values being 0.05 +/- 0.01 microM for PK-aerobic and 1.3 +/- 0.1 microM for PK-anoxic. In the presence of 1.0 mM alanine and 1.5 mM MgATP much higher concentrations of fructose 1,6-bisphosphate were required for activation of PK-anoxic (Ka = 5.2 +/- 0.4 microM) than for PK-aerobic (Ka = 0.02 +/- 0.01 microM). Variations in pH over the range likely occurring in vivo during anaerobiosis caused no significant additional kinetic differences between the two enzyme forms. The dissimilarity in kinetic properties of PK-aerobic and PK-anoxic indicate that red muscle PK activity is probably strongly depressed in vivo during anoxia stress.

Phosphorylation in vivo of red-muscle pyruvate kinase from the channelled whelk, Busycotypus canaliculatum, in response to anoxic stress.

That red muscle pyruvate kinase from anoxic Busycotypus canaliculatum (PK-anoxic) is a phosphoprotein was demonstrated by the anoxia-dependent, in vivo, covalent incorporation of injected [32P]orthophosphate into the enzyme molecule. Specificity in labelling of PK-anoxic was strongly suggested by: (a) coincidental elution of pyruvate kinase activity and radioactivity following chromatography of purified PK-anoxic on Sepharose CL-6B, and (b) comigration of the area containing [32P]phosphate and Coomassie-Blue-staining protein following SDS-polyacrylamide gel electrophoresis of homogenous PK-anoxic. The [32P]phosphate content of the enzyme was calculated to be 7.3 mol phosphate/mol enzyme (233 kDa, 180 units/mg protein). Evidence for the reversibility of this phosphorylation was provided by the consistent kinetic similarities between purified red muscle pyruvate kinase from aerobic animals (PK-aerobic) and homogenous, unlabelled, alkaline phosphatase treated PK-anoxic. Comparison of the electrophoretic mobilities of products derived from acid hydrolysis of purified 32P-labelled PK-anoxic with authentic substances suggest the presence of an O-phospho-L-threonine residue in the protein. That this residue plays a probable role in an interconversion mechanism was suggested by the lack of phosphate exchange of homogenous 32P-labelled PK-anoxic in the presence of all substrates. A possible role of protein phosphorylation as a mechanism for the overall control of molluscan anaerobic metabolism is suggested.

Buffering Capacities of the Tissues of Marine Molluscs

Buffering capacities (β) (measured in slykes = micromoles of base required to titrate the pH of 1 g wet weight of tissue by one pH unit between pH 6 and pH 7) due to nonbicarbonate buffers were measured for the tissues of three marine molluscs, the oyster, Crassostrea virginica, the cherrystone clam, Mercenaria mercenaria, and the channeled whelk, Busycotypus canaliculatum. In general β was higher in muscle tissues (adductors, foot, heart, radular retractor) than in the gill and mantle, but hepatopancreas of the whelk had a β two- to threefold higher than that of any other whelk tissue. Selected tissues (gill of oyster, catch adductor of clam, heart and hepatopancreas of whelk) showed a significant increase in buffering capacity when tissues were isolated from animals held under anoxic conditions compared with control aerobic animals; however, no consistent effect of environmental anoxia was found, nor was buffering capacity related to the tissue levels of succinate accumulated during anaerobiosis. Tissues of oysters and clams showed a positive correlation between β and the activities of terminal glycolytic dehydrogenases, and white muscle-type tissues (adductors, foot) of all three species showed a positive correlation between β and total dehydrogenase activities (lactate, octopine, and alanopine/strombine dehydrogenases). Buffering capacity appears to be related to the tissue need for "burst" glycolytic energy production (functional anoxia) rather than to the capacity for long-term anaerobiosis (environmental anoxia).

Purification and properties of alanopine dehydrogenase isozymes from the channeled whelk, Busycotypus canaliculatum

1. 1. Three tissue specific isozymes of alanopine dehydrogenase (ADH: alanopine: NAD oxidoreductase) were purified to homogeneity from foot muscle, gill and hepatopancreas of the channeled whelk, Busycotypus canaliculatum. 2. 2. Muscle ADH showed properties, in particular the effect of decreasing pH in lowering apparent K m's for pyruvate and l-alanine, which appear to gear this isozyme for alanopine synthesis as an end product of anaerobic glycolysis. 3. 3. The hepatopancreas isozyme, with a high affinity for meso-alanopine and NAD, appears suited for a role in alanopine oxidation in vivo. 4. 4. The properties of gill ADH are intermediate between those of the other two forms.

Tissue specific isozymes of alanopine dehydrogenase in the channeled whelk Busycotypus canaliculatum

The tissues of the channeled whelk Busycotypus canaliculatum displayed activities of three glycolytic dehydrogenases, alanopine dehydrogenase (ADH), octopine dehydrogenase (ODH), and lactate dehydrogenase (LDH). ADH and ODH were present in all seven tissues (hepatopancreas, gill, kidney, and mantle, ventricle, foot, and proboscis muscles) tested. ADH was the major cytosolic dehydrogenase activity in all tissues except proboscis, while ODH was present in high activities only in the four muscular tissues. Significant LDH activity occurred only in the two muscles which perform sustained work, ventricle, and proboscis.Tissue specific isozymes of ADH were identified. Three forms, specific for hepatopancreas, gill–kidney, and muscle tissues, were separable by isoelectrofocusing (pI's 5.69, 5.58, and 5.93, respectively), chromatofocusing, and polyacrylamide gel electrophoresis. The three isozymes differed kinetically showing significant differences in apparent Km's for L-alanine (e.g., 8.84 ± 0.03, 10.64 ± 0.40, and 13.12 ± 0.65 mM for hepatopancreas, ventricle, and gill, respectively) and even greater differences in apparent Km's for glycine (619 ± 55, 1412 ± 115, and 2542 ± 230 mM for the above three tissues, respectively). The ratios Km(gly)/Km(ala) averaged 70, 192, and 132 for the three isozymes, hepatopancreas, gill–kidney and muscle, respectively.The physiological functions of ADH isozymes in the whelk may be similar to those of the M and H isozymes of LDH in vertebrates or the muscle and brain specific isozymes of ODH in cephalopod molluscs.