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

1. 我们从无静脉条纹蛇的血清中鉴定出一个与中国马马鱼PLIα序列同源性约70%但不具有抑制活性的PLIα样蛋白，并克隆了其cDNA。我们构建了PLIα样蛋白与中国mamushi PLIα之间的重组嵌合蛋白，发现中国mamushi PLIα的13 ~ 36个残基序列对PLA_<2->的结合和抑制具有重要意义。此外，我们发现氨基酸残基26、28和29在该区域尤为重要。另一方面，为了确定PLIα的三级结构，我们寻找了从中国马木鱼血清中纯化的PLIα的最佳结晶条件，并获得了其针状晶体。然而，x射线衍射图像尚未得到。我们从条纹蛇的血清中鉴定出特异性抑制ii基碱性蛋白的PLIβ，并对其进行纯化和cDNA克隆。另外，我们正在尝试建立中国马穆氏PLIβ的大肠杆菌表达系统。另一方面，我们寻找了从中国马木鱼血清中纯化PLIβ的最佳结晶条件，但到目前为止还没有获得适合x射线结晶学的晶体3。我们正在研究PLIγ能否在不丧失抑制活性的情况下，从中国马木鱼PLIγ分离的两个亚基中重建出来。另一方面，我们寻找了从中国马木鱼血清中纯化PLIγ的最佳结晶条件，但此时我们还没有获得适合x射线结晶学的晶体。

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.