1. Alanopine dehydrogenase from the foot muscle of the common periwinkle, Littorina littorea was purified to homogeneity using a combination of ammonium sulphate fractionation, gel filtration and chromatofocusing. 2. The enzyme has a molecular weight of 42,200±500 and is a monomer. 3. l-Alanine and pyruvate are the preferred substrates. Alternate amino acids (glycine>l-α-aminobutyrate>l-serine>l-cysteine) are used at rates less than 37% of enzyme activity with l-alanine. Alternate keto acids used include oxaloacetate, α-ketobutyrate and glyoxylate. The enzyme is specific for meso-alanopine in the reverse direction;d-strombine is not oxidized. 4. Apparent Km's for both pyruvate and l-alanine decrease with increasing co-substrate (l-alanine or pyruvate) concentration or with decresing pH. 5. Absolute Km's for pyruvate and l-alanine are 0.17±0.02 and 14.9±0.85 mM at pH 6.5 rising to 0.26±0.01 and 23.8±0.52 mM at pH 7.5, respectively. Apparent Km's for meso-alanopine are 6.5 mM at pH 6.5 and 50 mM at pH 8.5 while apparent Km's for NADH (9±0.1 μM) and NAD+ (0.18±0.03 mM) are pH independent. 6. Substrate inhibition by pyruvate (I50=8 mM) and l-alanine (I50=450-550 mM) occurs at saturating co-substrate levels while NAD+ (Ki=0.16±0.012 mM) and meso-alanopine (Ki=35±0.4 mM) are product inhibitors of the forward reaction. ATP and ADP are competitive inhibitors with respect to NADH while l-lactate, d-strombine and succinate inhibit with respect to pyruvate and l-alanine. 7. The kinetic properties of alanopine dehydrogenase favour enzyme function in cytoplasmic redox balance during anoxia stress in this intertidal gastropod. In particular the effects of rising cosubstrate levels (pyruvate and alanine are products of glycolysis) and decreasing pH (occurring during anoxia) on enzyme apparent Km's for substrates would favour alanopine accumulation as a product of anaerobic glycolysis.

Journal of Comparative Physiology B
Department of Biology

Plaxton, W.C. (William C.), & Storey, K. (1982). Alanopine dehydrogenase: Purification and characterization of the enzyme from Littorina littorea foot muscle. Journal of Comparative Physiology B, 149(1), 57–65. doi:10.1007/BF00735715