Michaud D, Cantin L, Vrain T C
Pacific Agriculture Research Centre, Agriculture and Agri-Food Canada, British Columbia.
Arch Biochem Biophys. 1995 Oct 1;322(2):469-74. doi: 10.1006/abbi.1995.1490.
The biochemical interactions between digestive proteinases of the Coleoptera pest black vine weevil (Otiorynchus sulcatus) and two plant cysteine proteinase inhibitors, oryzacystatin I (OCI) and oryzacystatin II (OCII), were assessed using gelatin-polyacrylamide gel electrophoresis, OCI-affinity chromatography, and recombinant forms of the two plant inhibitors. The insect proteinases were resolved in gelatin-containing polyacrylamide gels as five major bands, only three of them being totally or partially inactivated by OCI and OCII. The maximal inhibitory effect of both OCs at pH 5.0 was estimated at 40% and the inhibition was stable with time despite the presence of OC-insensitive proteases, indicating the stability of the OCI and OCII effects. After removing OC-sensitive proteinases from the insect crude extract by OCI-affinity chromatography, the effects of the insect cystatin-insensitive proteases on the structural integrity of the free OCs were analyzed. While OCI remained stable, OCII was subjected to limited proteolysis leading to its gradual transformation into a approximately 10.5-kDa unstable intermediate, OCIIi. As shown by the degradation pattern of a glutathione S-transferase (GST)/OCII fusion protein, the appearance of OCIIi resulted from the C-terminal truncation of OCII. Either free or linked to GST, OCIIi was as active against papain and human cathepsin H as OCII, and the initial specificities of the inhibitor for these two cysteine proteinases were conserved after cleavage. Although these observations indicate the high conformational stability of OCII near its active (inhibitory) site, they also suggest a general conformational destabilization of this inhibitor following its initial cleavage, subsequently leading to its complete hydrolysis. This apparent susceptibility of OCII to proteolytic cleavage by the insect proteinases could have major implications when planning the use of this plant cystatin for insect pest control.
利用明胶 - 聚丙烯酰胺凝胶电泳、OCI亲和层析以及两种植物抑制剂的重组形式，评估了鞘翅目害虫黑葡萄象甲（Otiorynchus sulcatus）的消化蛋白酶与两种植物半胱氨酸蛋白酶抑制剂——水稻半胱氨酸蛋白酶抑制剂I（OCI）和水稻半胱氨酸蛋白酶抑制剂II（OCII）之间的生化相互作用。昆虫蛋白酶在含明胶的聚丙烯酰胺凝胶中分离为五条主要条带，其中只有三条被OCI和OCII完全或部分失活。两种OC在pH 5.0时的最大抑制作用估计为40%，尽管存在对OC不敏感的蛋白酶，但随着时间的推移抑制作用稳定，表明OCI和OCII作用的稳定性。通过OCI亲和层析从昆虫粗提物中去除OC敏感蛋白酶后，分析了昆虫胱抑素不敏感蛋白酶对游离OC结构完整性的影响。虽然OCI保持稳定，但OCII经历了有限的蛋白水解作用，导致其逐渐转化为约10.5 kDa不稳定中间体OCIIi。如谷胱甘肽S - 转移酶（GST）/OCII融合蛋白的降解模式所示，OCIIi的出现是由于OCII的C端截短。无论是游离的还是与GST相连，OCIIi对木瓜蛋白酶和人组织蛋白酶H的活性与OCII相同，并且在切割后抑制剂对这两种半胱氨酸蛋白酶的初始特异性得以保留。尽管这些观察结果表明OCII在其活性（抑制）位点附近具有高构象稳定性，但它们也表明该抑制剂在初始切割后普遍存在构象不稳定，随后导致其完全水解。当计划将这种植物胱抑素用于害虫防治时，OCII对昆虫蛋白酶蛋白水解切割的这种明显敏感性可能具有重大意义。
