PRESTON, R.L.*; Preston, E.J.D.; Middaugh, D.R.: Enzymatic Racemization of Amino Acids in Marine Invertebrate Tissues

Previously we showed that D-amino acids are present in the cellular free amino acid pools of diverse groups of marine invertebrates. Using a coupled enzyme assay employing D-amino acid oxidase or chiral HPLC, we surveyed over 130 species of marine invertebrates in 12 phyla. About 40% of these species in 10 phyla showed detectable levels of D-amino acids. In some cases, very high concentrations were observed, e.g. gill tissue of the soft-shelled clam, Mya arenaria, contained 63 mM D-alanine. D-Aspartate, D-glutamate, D-serine and D-alanine were detected in other species. We also showed that ¹⁴C-D-alanine was absorbed via membrane transport processes and metabolized. Transport occurred via low affinity L-amino acid preferring pathways or by D-stereoselective transport systems. The major pathway for D-alanine metabolism involved racemization. Using a modification of the coupled enzyme assay and chiral HPLC, we detected alanine racemase activity in 15 species of marine invertebrate (5 phyla). Analysis of partially purified racemase from Mya gill revealed this enzyme was relatively specific for alanine (Km 9 mM). We screened other species for alanine and serine racemase activity. Some species expressed only serine racemase activity (e.g. Limulus polyphemus) or only alanine racemase activity (e.g. Lunatia heros). Other species expressed both serine and alanine racemase activity (e.g. Asterias forbesi). We currently are purifying alanine racemase from Mya and have tentatively identified the alanine racemase gene. These data support the hypothesis that D-amino acids have significant biological roles in marine invertebrate tissues. Accumulating evidence suggests that D-amino acids may function as neuromodulators, as precursors for endogenous antibiotics, may have a role in osmoregulation and provide a contribution to the nutrition of marine invertebrates.