| Research Abstract |
Acid cysteine proteinase in the eggs of the silkmoth, Bombyx mori, exists as an inactive proenzyme and is glycosylated. This 47-kDa pro-BCP^1 zymogen molecule can be processed in vitro into an enzymatically active 39-kDa BCP molecule (Takahashi et al., 1993, J.Biochem (Tokyo), 97,701-701 ; Yamamoto et al., 1994, J.Biochem. (Tokyo), 116,1330-1335). In this current study, the maximum rate of processing in vitro was achieved at approximately pH 4.0, at a temperature of 37ﾟC under reducing conditins. Precursor processing was inhibited by a variety of cysteine proteinase inhibitors, but not by EDTA and pepstatin. The rate of conversion was not affected by increasing concentrations of pro-BCP.We prepared immobilized BCP bound to AH-Sepharose and examined the activation. Immobilized pro-BCP was autolysed, although the rate of processing was slow, indicating that the reactin might be an intramolecular one. Kinetic experiments suggest that the mechanism is likely to involve a stepwise reaction, in which pro-BCP is converted to an active enzyme through intermediate forms releasing small peptides stepwise. The first major intermediate might be generated by removal of the amino terminal 27 residues from pro-BCP^2 (Val_<-105>-Glu_<-78>). Then two peptides (Asp_<-77>-Ser_<-44'> or Trp_<-43> and Trp_<-43> or Trp_<-42>-Ile_<-1>) might be esequentially released from the NH_2-terminal region and the enzyme is finally processed to its active form (39-kDa).
The results suggest that autocatalytic cleavage (intramolecular) is a major processing step in the early stage of pro-BCP activatin.
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