PRODUCTION OF SERINE PROTEASES BY THE OYSTER PATHOGEN PEUKINSUS-MARINUS (APICOMPLEXA) IN-VITRO

Analysis of the cell-free supernatants of Perkinsus marinus cultures b y sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and silver staining revealed the presence of as many as 17 bands rangi ng in molecular weight from 239 to 32 kDa. These bands were not presen t in un-inoculated medium. Moreover, P. marinus produces extracellular proteins that possess proteolytic activities; the cell-free supernata nts of P. marinus cultures could digest a variety of proteins includin g gelatin, casein, fibronectin and laminin. Oyster plasma was also dig ested by cell-free culture supernatants. The proteolytic activity in c ell-free culture supernatants was detected 24 h post-inoculation, whil e no proteolytic activity could be detected in cell lysates. The prote olytic activities were characterized using substrate-impregnated sodiu m dodecylsulfate-polyacrylamide gels and had approximate molecular wei ghts ranging from 55 to 35 kDa. The proteolytic activity of cell-free culture supernatants was inhibited by the serine protease inhibitors p henylmethylsulphonyl fluoride, 3,4-dichloroisocoumarin and soybean try psin inhibitor. In contrast, inhibitors (i.e. ns-epoxysucciny-1-leucyl amido(4-guanidino)-butane, 1,10-phenanthroline, captopril, ethylenedia minetetracetic acid, pepstatin A or diazoacetyl-DL-norleucine methyl e ster) from the other three classes of proteases had no effect. It was concluded that the P. marinus proteases in cell-free culture supernata nts are serine proteases.