A trypsin-like serine proteinase from plasmodial membrane of Physarum polycephalum

A trypsin-like serine proteinase was found in a plasma membrane fraction from the plasmodia of Physarum polycephalum, It was solubilized from the plasma membrane fraction with 1% (w/v) Triton X-100 and purified by a series of column chromatographic steps followed by polyacrylamide gel electrophoresis (PAGE). The molecular mass of the enzyme was estimated to be 130 kDa by SDS-PAGE under non-reducing conditions. This enzyme exhibited maximal activity at pH 9 and at 35 degrees C with the substrate Boc-Phe-Ser-Arg-MCA, and was sensitive to various serine proteinase inhibitors and trypsin inhibitors. The enzyme was significantly labeled with [H-3] diisopropylfluorophosphate (DFP); however, a clear decrease in the amount of the enzyme bound-[H-3]DFP was observed in the presence of p-nitrophenyl-p'-guanidinobenzoate (NPGB), an active site titrant for trypsin. The activity was completely inhibited in the presence of EDTA or EGTA, but the metal-depleted enzyme was reactivated by the addition of Ca2+. Moreover, this enzyme was capable of cleaving several synthetic peptides at the carboxyl side of arginine, but did not work on synthetic substrates for chymotrypsin, elastase and amino-peptidase, The enzyme showed an affinity to Con A, indicating that it contains a carbohydrate moiety. Inhibition of the activity by a serine proteinase inhibitor resulted in cessation of the DNA synthesis in growing plasmodia without loss of the protein synthesis. Thus, this trypsin-like serine proteinase may have a regulatory role in the growth and development of the slime mould.