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目的 建立SAH水解酶体外抗病毒筛选模型。方法 由大鼠肝经分级硫酸铵沉淀及各种柱层析 (DEAE5 2 ,羟基磷灰石及SephadexG 10 0 )分离 ,纯化SAH水解酶。用同位素标记底物 ,以合成方向建立酶活性测定及抑制剂筛选方法。结果 纯化的SAH水解酶在SDS PAGE电泳上呈单一蛋白带 ,表现相对分子质量为 45 0 0 0 ,其底物腺苷的米式常数值为 (6 32± 0 17) μmol L。已知SAH水解酶抑制剂 S DHPA的IC50 为 7 6 μmol L。用消旋及位置异构体DHPA证实S DHPA抑制SAH水解酶活性的结构特异性很强。用此模型筛选不同结构化合物 42个 ,未发现抑制活性强于S DHPA的化合物。结论 由大鼠肝提纯SAH水解酶 ,并建立了体外模型 ,可用于抗病毒化合物筛选及酶抑制剂动力学研究
Objective To establish an in vitro antiviral screening model of SAH hydrolase. Methods SAH hydrolase was purified from rat liver by graded ammonium sulphate precipitation and various column chromatography (DEAE5 2, hydroxyapatite and Sephadex G 10 0). Substrates are labeled with isotopes to establish enzymatic activity assays and inhibitor screening approaches in a synthetic direction. Results The purified SAH hydrolase showed a single protein band on SDS PAGE electrophoresis. The relative molecular mass was 45 0 0 0, and the substrate constant of adenosine was (6 32 ± 0 17) μmol L. The IC50 of SAH hydrolase inhibitor S DHPA is known to be 76 μmol L. The structural specificity of S DHPA for inhibiting SAH hydrolase activity was demonstrated using the racemic and positional isomer DHPA. Forty-two compounds with different structures were screened by this model. No compounds with stronger inhibitory activity than S DHPA were found. Conclusion SAH hydrolase was purified from rat liver and an in vitro model was established for the screening of antiviral compounds and the kinetics of enzyme inhibitor