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Inhibition Mechanism of a Novel Phospholipase A_2 Inhibitor from the Blood of the Venomous Snakes

毒蛇血磷脂酶A_2新型抑制剂的抑制机制

基本信息

批准号：
07672400
负责人：
INOUE Seiji
金额：
$ 1.47万
依托单位：
Osaka University of Pharmaceutical Sciences
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1996
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07672400/
关键词：
Phospholipase A_2 Inhibitor Venomous snake Amino acid sequence Inhibition mechanism Blood cDNA

项目摘要

1. Purification of novel phospholipase A_2 inhibitors from the blood plasma of Chinese mamushiWe have purified three distinct types of phospholipase A_2 inhibitors (PLIalpha, PLIbeta, and PLIgamma) from the blood plasma of Chinese mamushi. PLIalpha was found to be CRD-like inhibitor that we had already reported to inhibit selectively the group-II acidic PLA_2s. PLIbeta and PLIgamma were novel inhibitors purified in the present study. The former inhibited selectively the group-II basic PLA_2s, while the latter inhibited equally all of the PLA_2s including group-I PLA_2s, group-II PLA_2s, and honey bee PLA_2 (group-III).2. cDNA cloning of Chinese mamushi PLA_2 inhibitorsWe have constructed cDNA library for the liver mRNA of Chinese mamushi, cloned cDNA for PLIalpha, PLIbeta, and PLIgamma, and determined their nucleotide sequences. As a results, PLIgamma was found to be the same type of the inhibitors that had been already purified from Thailand cobra plasma and sea snake plasma, having the similar pattern of cysteine residues to those of the proteins belonging to Ly-6 superfamily. On the other hand, PLIbeta was found to have high sequence homology to leucine-rich alpha_2 glycoprotein from human sera.3. Interaction between PLA_2 and inhibitorOne molecule of PLIbeta was composed of three identical subunits and it bound three PLA_2 molecules. From the comparisons of the specificity of PLIbeta, some amino acid residues located in or close to the interfacial binding surface of the group-II PLA_2 were suggested to be involved in the selective binding to PLIbeta. On the other hand, the residues located in or close to the Ca^<2+> binding loop of PLA_2 were suggested to be involved in the binding to PLIgamma.4. Purification of PLA_2 inhibitor from the sera of Elaphe quadrivirgataPLA_2 inhibitors were found to be present in the serum of nonvenomous snakes, Elaphe quadrivirgata, and were purified. The purified inhibitor was found to be the same type of PLIgamma.

1.从中华猛犸象血浆中纯化新型磷脂酶A_2抑制剂我们从中华猛犸象血浆中纯化了三种不同类型的磷脂酶A_2抑制剂（PLIalpha、PLIbeta和PLIgamma）。α是一种CRD样抑制剂，我们已经报道过它选择性地抑制Ⅱ型酸性PLA_2。在本研究中纯化的β-淀粉样蛋白和γ-淀粉样蛋白是新的抑制剂。前者选择性地抑制Ⅱ型碱性PLA_2，而后者对Ⅰ型PLA_2、Ⅱ型PLA_2和蜜蜂型PLA_2（Ⅲ型）的抑制作用相同。本研究构建了中国马蚊PLA_2基因cDNA文库，克隆了PLA_2基因的cDNA，并测定了其核苷酸序列。结果发现，Ly-γ与已经从泰国眼镜蛇血浆和海蛇血浆中纯化的抑制剂是相同类型的，具有与属于Ly-6超家族的蛋白质相似的半胱氨酸残基模式。另一方面，发现β-淀粉样蛋白与来自人血清的富含亮氨酸的α_2糖蛋白具有高度的序列同源性。PLA_2与β-内酰胺酶的相互作用一个β-内酰胺酶分子由三个相同的亚基组成，它与三个PLA_2分子结合。通过对β-内酰胺酶特异性的比较，推测Ⅱ型PLA_2界面结合表面附近的氨基酸残基可能参与β-内酰胺酶的选择性结合。另一方面，位于PLA_2的Ca^2+结合环中或其附近的残基被认为参与了PLA_2与γ的结合。从四纹锦蛇血清中纯化PLA_2抑制剂在四纹锦蛇血清中发现PLA_2抑制剂，并进行了纯化。纯化的抑制剂被发现是相同类型的γ射线。