Purification and properties of the malic enzyme and the malate dehydrogenases from the gill tissue of the ribbed mussel (Modiolus demissus)

To assess the role of dicarboxylic acid metabolism in the synthesis of alanine and other compounds by the ribbed mussel under conditions of hyperosmotic and anaerobic stress, the cytosolic and mitochondrial malate dehydrogenases and the mitochondrial malic enzyme from this organism were purified and their kinetics studied. Both the cytosolic and mitochondrial malate dehydrogenases were found to be dimers with native M[subscript] r = 60,000. One cMDH allele (designated slow) was found to predominate in populations from Cape Cod, MA. Similar low K[subscript] m\u27s were observed for purified preparations of the slow/slow homodimer (cMDH) and for the purified mitochondrial MDH (mMDH) for the substrates NAD and NADH under all experimental conditions. The mMDH had lower apparent K[subscript] m\u27s for oxaloacetate and higher apparent K[subscript] m\u27s for malate under all experimental conditions. Both forms exhibited substrate inhibition by oxaloacetate and malate with the mMDH more sensitive as assessed by the lower apparent K[subscript] i\u27s for these substrates. The mMDH was more sensitive to inhibition by sodium chloride, and lithium chloride than the cMDH. Alanine and sodium acetate were inhibitory to the cMDH, whereas the mitochondrial form was insensitive to inhibition by these compounds. The mMDH was more sensitive to inhibition by the malate analogue hydroxymalonate than the cMDH. The cMDH was more sensitive to inhibition by ATP than the mMDH. The mitochondrial malic enzyme (ME) had high affinity for metal ions. Mg[superscript]2+ (K[subscript]0.5 140 micromolar) and Co[superscript]2+ (K[subscript]0.5 2 picomolar) were able to restore only 60% of the Mn[superscript]2+ (K[subscript]0.5 18 picomolar) dependent activity. Only limited reactivity was found when NAD was substituted for NADP. Each of the several salts tested (sodium chloride, potassium chloride, lithium chloride, and sodium acetate), hydroxymalonate, and fumarate were inhibitory. Product inhibition patterns indicated that this enzyme operates according to a random reaction mechanism. This ME was a tetramer with native M[subscript] r 265,000. The co-localization of these activities with aspartate aminotransferase and the alanine aminotransferase may account for the appearance in alanine of aspartate derived label during hyperosmotic stress and anaerobic stress