Analysis of the effects of Perkinsus marinus proteases on plasma proteins of the eastern oyster (Crassostrea virginica) and the Pacific oyster (Crassostrea gigas)

We employed two in vitro buffer systems to determine the potential pathogen ic effects of Perkinsus marinus serine proteases on the plasma proteins of the eastern oyster (Crassostrea virginica) and the Pacific oyster (Crassost rea gigas). Specifically, this study characterized the oyster plasma protei n targets of P. marinus proteases. Additionally, protease-specific inhibito ry activity was revealed upon comparison of artificial (PBS) and endogenous (plasma-based) diluents employed during protease digestions. It was found that a C. virginica plasma protein of approximately 35 kDa was eliminated w hen a standard buffer (PBS) was used as a diluent; however this protein was preserved when a low-molecular-weight, plasma-based, diluent was used. The results strongly indicate that low-molecular-weight inhibitors of P. marin us proteases are present in oyster plasma. A control (nonparasitic) serine protease, alpha-chymotrypsin, was employed to ascertain the specificity of the protease inhibitors. Although alpha-chymotrypsin possesses ample proteo lytic activity for C. virginica plasma proteins, the anti-proteases could s pecifically inhibit only P. marinus proteases. Such specificity of anti-pro tease activity is not uncommon among low-molecular-weight serine proteases. The hemolymph target protein was isolated by 2D electrophoresis and isoele ctrically isolated for further characterization by N-terminal amino acid se quencing. (C) 1999 Academic Press.