BOMBYX CYSTEINE PROTEINASE (BCP) : X-RAY DIFFRACTION ANALYSIS.

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In the silkmoth eggs, a large amount of inactive pro-cysteine protease accumulates. BCP(47kDa) is processed in vitro into an enzymatically active 39-kDa molecule(M-form). Similar active enzyme is also found in developing silkmoth eggs, indicating that the activation of this enzyme is a key step in yolk protein degradation. In order to know the detailed mechanism of activation, we examined the structure of proBCP using X-ray crystallography, circular dichroiam and high voltage electron microscopy, as well as kinetic experiments. Gel filtration and SDS-PAGE analysis suggest that proBCP exist as an octameric structure. In this study we directly demonstrated the octameric structure of proBCP by high voltage electron microscopy. Brief exposure of proBCP to acidic conditions, it was cornverted to a monomeric structure. If it is exposed to acidic conditions for a longer period, limited proteolysis occurs at N-terminal region and converted to 39-kDa active form. Kinetic experiments suggest that the reaction might be an autocatalytic intramolecular reaction, in which monomeric pro-BCP is converted to an active enzyme through intermediate forms, X & Y, releasing small peptides. This is the major processing in the early stage of pro-BCP activation. In addition, we crysalized pro-BCP and, to confirm above theory. Furthermore, we found a novel cysteine protease specific inhibitors, cloned cDNA and amino acid sequense of it was determined.