A SERINE-PROTEASE ZYMOGEN IN INSECT PLASMA - PURIFICATION AND ACTIVATION BY MICROBIAL CELL-WALL COMPONENTS

A protease zymogen present in the plasma fraction of the hemolymph of silkworm, Bombyx mori, was purified to homogeneity as judged by SDS/PA GE and IEF/PAGE. An activating system for the zymogen was also isolate d from the plasma fraction and was shown to be triggered by zymosan (y east cell wall polysaccharide containing beta-1,3-glucan) or peptidogl ycan. Using this system, the purified zymogen was activated and the ac tive enzyme was purified to homogeneity. The physiological function of the zymogen or its active form is not yet known, but the active form was shown to have narrower substrate specificity than trypsin. Among 3 3 peptide derivatives examined, Boc-Gln-Arg-Arg-NH-Mec and Boc-Val-Pro -Arg-NH-Mec (Boc = tert-butoxycarbonyl, NH-Mec = 4-methylcoumaryl-7-am ide) were the best and the second best substrates, respectively. The p urified zymogen was determined to be a 39-kDa protein consisting of a single polypeptide. The active form of the zymogen was labeled with [H -3]diisopropylfluorophosphate and was completely inactivated by (p-ami dinophenyl)methanesulfonyl fluoride. The molecular mass of the [H-3]-l abeled enzyme was determined to be 38 kDa in SDS/PAGE under reducing c onditions. These results indicate that the 39-kDa protein purified in the present study is a zymogen of a serine-type protease and that the activation of the zymogen occurs by limited proteolysis.