Jf. Lapeyre et al., PRODUCTION OF SERINE PROTEASES BY THE OYSTER PATHOGEN PEUKINSUS-MARINUS (APICOMPLEXA) IN-VITRO, The Journal of eukaryotic microbiology, 42(5), 1995, pp. 544-551
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.