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

在蚕卵中，积累了大量的无活性的半胱氨酸蛋白酶原。BCP（47 kDa）在体外被加工成具有酶活性的39 kDa分子（M型）。在发育中的蚕卵中也发现了类似的活性酶，表明该酶的激活是卵黄蛋白降解的关键步骤。为了了解活化的详细机制，我们用X射线晶体学、圆二色谱和高压电子显微镜以及动力学实验研究了proBCP的结构。凝胶过滤和SDS-PAGE分析表明proBCP以八聚体结构存在。在这项研究中，我们直接证明了八聚体结构的proBCP的高压电子显微镜。proBCP短暂暴露于酸性条件下，转变为单体结构。如果暴露于酸性条件较长时间，则在N-末端区域发生有限的蛋白水解，并转化为39-kDa的活性形式。动力学实验表明，该反应可能是一个自催化的分子内反应，其中单体pro-BCP通过中间形式，X和Y，释放小肽转化为活性酶。这是pro-BCP活化早期的主要过程。此外，我们还对pro-BCP进行了晶体化，证实了上述理论。此外，我们还发现了一种新的半胱氨酸蛋白酶特异性抑制剂，并对其cDNA进行了克隆和氨基酸序列测定。

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.