| Co-Investigator(Kenkyū-buntansha) |
KIHARA Hiroshi Takara Shuzo Co., Senior Scientist, バイオ研究所, 次長
HATTORI Shosaku Tokyo Univ., Inst.of Med.Sci., Assis.Prof., 医科学研究所, 講師 (00164864)
OGAWA Tomohisa Kyushu Univ., Fac.of Sci., Res.Assoc., 理学部, 助手 (80240901)
SHIMOHIGASHI Yasuyuki Kyushu Univ., Fac.of Sci., Assoc.Professor, 理学部, 助教授 (00211293)
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| Research Abstract |
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
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