Purification and properties of alanopine dehydrogenase from the adductor muscle of the oyster, Crassostrea gigas (Mollusca, Bivalvia).

Abstract

Alanopine dehydrogenase (alanopine: NAD oxidoreductase) was purified from the adductor muscle of the Japanese oyster, Crassostrea gigas Thunberg. This enzyme catalyzes the reductive imination between pyruvate and alanine, or glycine, utilizing NADH as coenzyme, producing 2,2′-iminodipropionic acid (alanopine), or 2-methyliminodiacetic acid (strombine). The enzyme can be readily purified to a specific activity of 700 units/mg protein. The molecular weight was 47000 ± 4000 when measured by gel filtration on Sephadex G-200, or by electrophoresis on 10% polyacrylamide gels in the presence of 1% sodium dodecyl sulphate. The latter value was unaltered by treatment with 8 M guanidine hydrochloride, suggesting that alanopine dehydrogenase is composed of it single polypeptide. Of the keto acids tested for activity with the enzyme, only pyruvate and 2-oxobutyrate reacted significantly as substrates; among the amino acids tested, highest activities occurred with alanine and glycine, but 2-aminobutyrate and serine also reacted as substrates. Apparent Km values obtained for the substrates are 0.01 mM, 0.45 mM, and 100 mM for NADH, pyruvate and alanine (or glycine) respectively when measured at approximately saturating concentrations of cosubstrates at pH 7.0. The product, NAD+ was bound to be a competitive inhibitor with respect to NADH, and the low apparent Ki for NAD+ (0.22 mM) at pH 7.0 may indicate that the enzyme is closely regulated by the redox status of the cell. Several metabolites were tested for possible effects on the enzyme; of these ATP, ADP, AMP, succinate and 2-oxoglutarate were inhibitors. The adenylates inhibited competitively with NADH. The inhibition by 2-oxoglutarate was uncompetitive with respect to alanine and pyruvate, in both cases the apparent Ki value being 0.7 mM at pH 7.0. Succinate inhibition was non-competitive with respect to alanine and pyruvate, the apparent Ki values being strongly dependent on pH (22.0 mM, 6.5 mM, and 1.8 mM at pH 7.5, 7.0, and 6.5 respectively). The physiological role of the enzyme during anaerobiosis is discussed.