METABOLIC ENZYMES AND REGULATION OF FREE D-AMINO ACIDS IN INVERTEBRATES

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Alanine racemase was isolated from the muscle of black-tiger prawn. Partial amino acid sequences were obtained from the peptide fragments of purified enzyme. These sequences showed similarity with the enzyme from some bacteria but the similarities were below 40%. The prawn enzyme was considered to be rather different from bacterial counterpart in its structure and properties. Antibody was prepared on the basis of these partial sequences and used for the examination of subcellular distribution of the enzyme and the screening of cDNA library.Distribution of D-amino acid and D-aspartate oxidases in fish was species specific and considered to be related to food habits. The activity of D-amino acid oxidase increased in intestine, hepatopancreas, and kidney of carp with the administration of D-alanine, suggesting the enzyme is an inducible one. No enzyme induction occurred in D-aspartate oxidase with the administration of D-aspartate or D-glutamate. Both enzyme activities were subcellularly … More distributed in peroxisomes as is the case in mammals.cDNA cloning of D-amino acid oxidase from carp hepatopancreas gave a PCR product of 801 bp encoding 267 amino acid residues. The amino acid sequence showed similarities of 58-59% to human and porcine one.In the tissues of crayfish, kuruma prawn, and hard clam, D-, L-alanine increased significantly with increasing environmental salinity, indicating that D-alanine is a major osmolyte responsible for intracellular isosmotic regulation in these invertebrates. After hypersalinity acclimation, crayfish received severe anoxia increased D-, L-alanine in muscle and hepatopancreas which declined during recovery after anoxia. From these data, D-, L-alanine were considered to be anaerobic end-products together with lactate in crayfish.In the present study, physiological roles of D-alanine in invertebrates were clarified and the partial amino acid sequences of alanine racemase were determined for the first time. It was also clarified that fish species having no D-alanine metabolized D-amino acids to corresponding α-keto acid with D-amino acid and D-aspartate oxidases in liver and kidney and detoxified D-amino acids. Less