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Structure and function of three types of phospholipase A_2 inhibitor s derived from the blood of venomous snakes

三种毒蛇血磷脂酶A_2抑制剂的结构与功能

基本信息

批准号：
12672137
负责人：
INOUE Seiji
金额：
$ 2.18万
依托单位：
Osaka University of pharmaceutical Sciences, Assistant
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2001
项目状态：
已结题

项目摘要

1. Structure and function of PLIαWe have identified a PLIα-like protein, which showed about 70% sequence homology to Chinese mamushi PLIα but showed no iuhibitory activity from the serum of the non-venorilous striated snake and cloned its cDNA. We have constructed the recombinant chiineric proteins between the PLIα-like protein and Chinese mamushi PLIα and found that the sequence from 13 to 36 residues of Chinese mamushi PLIα were important for the bindihg and inhibition of the PLA_<2->. Furthermore, we found that the amino acid residues 26, 28, and 29 were especially important among this region. On the other hand, in order to determine the tertiary structure of PLIα, we searched for the best crystallization conditions of PLIα purified from the Chinese mamushi, serum, and we could obtain its needle crystal. However, the X-ray diffiaction image is not obtained yet.2. Structure and function of PLIαWe have identified PLIβ, which specifically inhibit group-II basic protein, from the serum of the striated snake, purified it, and cloned its cDNA. Alternatively, we are tying to establish the E. coli expression system for Chinese mamushi PLIβ. On the other hand, we searched for the best crystallization conditions of PLIβ purified from Chinese mamushi, serum, but we could not obtain the crystals suitable for X-ray crystallography by this time3. Structure and function of PLIγWe are investigating whether PLIγ could be reconstructed from the separated two subunits of Chinese mamushi PLIγ Without a loss of the inhibitory activity. On the other hand, we searched, for the best crystallization conditions of PLIγ purified from Chinese mamushi, serum, but we could not obtain the crystals suitable for X-ray crystallography by this time.

1.PLIα的结构和功能我们克隆了一个与中国乳鼠PLIα同源性约为70%的蛋白，但从无毒蛇血清中没有显示抑制活性，并克隆了它的α。我们构建了PLI类α蛋白与中国乳鼠PLIα之间的重组蛋白，发现中国乳鼠PLIα的13~36个残基的序列对PLI的结合和抑制具有重要作用。此外，我们还发现，26、28和29位氨基酸残基在该区域中尤为重要。另一方面，为了确定磷脂酶Iα的三级结构，我们寻找了从中国马鹿血清中纯化的磷脂酶Iα的最佳结晶条件，得到了它的针状晶体。然而，目前还没有得到X射线衍射图。磷脂酶Iα的结构和功能我们从纹蛇血清中鉴定了特异性抑制II组碱性蛋白的磷脂酶Iβ，并对其进行了纯化和克隆。或者，我们正在努力建立中国乳鼠PLIβ的大肠杆菌表达系统。另一方面，我们寻找了从中国马鹿血清中纯化的磷脂酶Iβ的最佳结晶条件，但到目前为止还没有得到适合于X射线结晶学的晶体。磷脂酶Iγ的结构和功能我们正在研究能否在不损失抑制活性的情况下，从分离的两个亚基中重建中国乳鼠磷脂酶Iγ。另一方面，我们寻找了从中国马鹿血清中纯化的磷脂酶Iγ的最佳结晶条件，但到目前为止还没有得到适合于X射线结晶学的晶体。