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Production of Serine Proteases by the Oyster Pathogen Perkinsus marinus (Apicomplexa) In Vitro
Author(s) -
PEYRE JEROME F. LA,
SCHAFHAUSER DORIS Y.,
RIZKALLA ESAM H.,
FAISAL MOHAMED
Publication year - 1995
Publication title -
journal of eukaryotic microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.067
H-Index - 77
eISSN - 1550-7408
pISSN - 1066-5234
DOI - 10.1111/j.1550-7408.1995.tb05903.x
Subject(s) - biology , proteases , biochemistry , leupeptin , pepstatin , pmsf , polyacrylamide gel electrophoresis , serine protease , microbiology and biotechnology , serine , protease , gel electrophoresis , cell culture , enzyme , genetics
. Analysis of the cell‐free supernatants of Perkinsus marinus cultures by sodium dodecylsulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and silver staining revealed the presence of as many as 17 bands ranging in molecular weight from 239 to 32 kDa. These bands were not present in un‐inoculated medium. Moreover, P. marinus produces extracellular proteins that possess proteolytic activities; the cell‐free supernatants of P. marinus cultures could digest a variety of proteins including gelatin, casein, fibronectin and laminin. Oyster plasma was also digested by cell‐free culture supernatants. The proteolytic activity in cell‐free culture supernatants was detected 24 h post‐inoculation, while no proteolytic activity could be detected in cell lysates. The proteolytic activities were characterized using substrate‐impregnated sodium dodecylsulfate‐polyacrylamide gels and had approximate molecular weights ranging from 55 to 35 kDa. The proteolytic activity of cell‐free culture supernatants was inhibited by the serine protease inhibitors phenylmethylsulphonyl fluoride, 3,4‐dichloroisocoumarin and soybean trypsin inhibitor. In contrast, inhibitors (i.e. trans‐epoxysuccinyll‐leucylamido(4‐guanidino)‐butane, 1, 10‐phenanthroline, captopril, ethylenediaminetetracetic acid, pepstatin A or diazoacetyl‐DL‐norleucine methyl ester) from the other three classes of proteases had no effect. It was concluded that the P. marinus proteases in cell‐free culture supernatants are serine proteases.