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Protein Chemistry and Basic Research for Application of Inhibitors from Trimeresurus flavoviridis Serum against Its Venom Enzymes

黄绿竹叶青血清毒酶抑制剂的蛋白质化学及应用基础研究

基本信息

批准号：
03554021
负责人：
OHNO Motonori
金额：
$ 9.54万
依托单位：
KYUSHU UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Developmental Scientific Research (B)
财政年份：
1991
资助国家：
日本
起止时间：
1991 至 1993
项目状态：
已结题

项目摘要

The object of this study is to isolate proteins from Trimeresurus flavoviridis serum which are bound to its venom enzymes and meutralize their activities, and to investigate chemical properties of these inhibitors and to make basic study for application of these inhibitors to therapy of T.flavoviridis bite. Two lysine-49-phospholipases A_2(PLA_2)(BP I and BP II) which are involved abundantly in T.flavoviridis venom were found to have strong myolytic activity although they were lipolytically 1-2% as active as Asp-49-PLA_2, a component of the venom. At first, we intended to isolate protein from T.flavoviridis serum which is bound to BP I.Serum proteins precipitated at 50% saturation of ammonium sulfate were fractionated on DEAE-cellulose column and BP I-conjugated Sepharose 4B column. The bound proteins (sample A) were further fractionated by FPLC (Mono-Q) to give a major fraction (sample B). The bound protein was a glycoprotein of MW 26,000. which was reduced to 23,000 after N-glycanase … More digestion.Mixtures of BP I-binding protein (sample A or B) and BP I were injected into thigh of mice and creatine kinase (CK) levels released to blood circulaton were quantitated. Samples A and B depressed CK production in dose-dependent manner, indicating that BP I-binding protein represses muscle necrosis caused by BP I.Such observation suggests that BP I-binding protein could be utilized as a drug for therapy of T.flavoviridis bite. Partial(50%) amino acid sequence of BP I-binding protein has been determined. On the other hand, two zinc proteases which have caseinolytic and plasmin-like activity, respectively, were purified from T.flavovoridis venom. Purification of serum inhibitors against these proteases were made by DEAE-cellulose column and protease-conjugated column chromatographies followed by HPLC.Two proteins of MW 65,000 and 29,000, respectively, were obtained. The former, a glycoprotein, inhibited both caseinolytic and plasmin-like proteases. The N-terminal sequence of this protein was different from that of anti-hemorrhagic factor which is bound to hemorrhagic zinc protease which had previously been isolated from T.flavoviridis venom. Less

本研究的目的是从黄病毒Trimeresurus血清中分离出与黄病毒Trimeresurus毒液酶结合并调节其活性的蛋白，研究这些抑制剂的化学性质，为这些抑制剂在黄病毒t .叮咬治疗中的应用进行基础研究。两种赖氨酸-49-磷脂酶A_2(PLA_2)（BP I和BP II）在黄毒t .黄毒中大量存在，具有较强的溶肌活性，但其溶脂活性仅为p-49-PLA_2的1-2%。首先，我们计划从与BP i结合的黄颡鱼血清中分离蛋白质，在硫酸铵饱和度为50%时沉淀的血清蛋白在deae -纤维素柱和BP i偶联的Sepharose 4B柱上进行分离。结合蛋白（样品A）进一步用FPLC （Mono-Q）分离得到主组分（样品B）。结合蛋白为分子量为26000的糖蛋白。在n -聚糖酶的作用下减少到23000个将BP I结合蛋白（样品A或样品B）与BP I的混合物注射到小鼠大腿，并定量释放到血液循环中的肌酸激酶（CK）水平。样品A和样品B呈剂量依赖性地抑制CK的产生，说明BP i结合蛋白抑制BP i引起的肌肉坏死，提示BP i结合蛋白可作为治疗黄病毒锥虫咬伤的药物。BP i结合蛋白部分（50%）氨基酸序列已确定。另一方面，从黄毒蛇毒中分离得到两种具有溶酪蛋白和纤溶酶样活性的锌蛋白酶。采用deae -纤维素柱和蛋白酶偶联柱层析纯化这些蛋白酶的血清抑制剂，然后采用高效液相色谱法。得到两个分子量分别为65,000和29,000的蛋白。前者是一种糖蛋白，能抑制酪蛋白溶酶和纤溶酶样蛋白酶。该蛋白的n端序列与先前从黄毒霉毒素中分离到的与出血性锌蛋白酶结合的抗出血性因子的n端序列不同。少