Comparative enzymology and physiological role of D‐lactate dehydrogenase from the foot muscle of two gastropod molluscs

Abstract

AbstractD‐Lactate dehydrogenases from the foot muscles of the black abalone, Haliotis cracherodii, and the pulmonate snail, Helix aspersa, have been characterized. The D‐lactate dehydrogenase from H. cracherodii, which is found as a single, isoenzymic form, was purified to homogeneity. The enzyme in H. aspersa foot muscle is found as two distinct fast and slow migrating electrophoretic complexes of isoenzymes. The most abundant isoenzyme in the slow electrophoretic complex of H. aspersa was purified to homogeneity. The purified enzymes from the two organisms have similar physical and catalytic properties to one another and to other D‐lactate dehydrogenases, suggesting a close evolutionary relationship. Purified D‐lactate dehydrogenases from H. cracherodii and H. aspersa have molecular weights of approximately 80,000, as judged by meniscus depletion sedimentation equilibrium experiments, which correspond to a dimeric subunit organization. Kinetic studies show that the apparent Michaelis constant for pyruvate is 0.2–0.3 mM for the two purified enzymes as well as the partially purified fast isoenzymic complex from snail. Km values for D‐lactate are about ten times higher. Km values for α‐ketobutyrate are 4–10 mM for all three enzymes and Vmax is approximately 40–80% of that seen with pyruvate, suggesting that the enzymes may be physiologically important in dealing with α‐ketoacids in general. Km'S for NADH are all 10–20 μM, and those for NAD+ about 10‐fold greater. The most notable catalytic difference between the two enzymes is the presence and absence of substrate inhibition by pyruvate of the abalone and snail enzymes, respectively. This difference may reflect different physiological roles for the enzyme in the two organisms.