【摘要】 以胡杨愈伤组织为材料,用PEG3350/DextranT500构成的两相系统提取质膜微囊,研究质膜H+-AT-Pase的特性。结果显示:由6.3%PEG3350、6.3%DextranT500、KCl、磷酸缓冲液(pH7.8)和蔗糖构成的两相系统提取膜微囊的H+-ATPase活性分别被Na3VO4、KNO3、NaN3抑制了约75%、2.6%和1.3%。方向性检测显示原位膜微囊占提取质膜微囊的90%,翻转膜微囊仅占10%。去垢剂对质膜H+-ATPase活性的影响说明0.015%的TritonX-100和0.01%～0.1%的Brij58适用于测定质膜H+-ATPase活性。Lineweaver-Burk动力学分析该酶的Km值为0.65mmol·L-1,Vmax为37.59μmolPi·mg-1protein·h-1。研究结果表明:两相法提取的质膜微囊主要是正向密闭的膜微囊;胡杨愈伤组织质膜H+-ATPase的最适pH为6.5,最适温度为37℃左右。更多还原

【Abstract】 Plasma membrane vesicles from Populus euphratica callus were isolated and purified by two-phase partition composed of Dextran T-500 and PEG 3350,and the properties of plasma membrane H+-ATPase were investigated.The H+-ATPase activities in membrane partials from two-phase partition were inhibited about 75%,2.6% and 1.3% by Na3VO4,KNO3 and NaN3,respectively.90% of membrane vesicles were right-side out,and in-side out membrane vesicles were only 10%.Investigation of the effects of detergents on plasma membrane H+-ATPase activity indicated that 0.015% Triton X-100 and 0.01%~0.1% Brij 58 were suitable for assay of plasma membrane H+-ATPase activity.A conventional Lineweaver-Burk plot of plasma membrane H+-ATPase yielded a Km value of 0.65 mmol/L and a Vmax value of 37.59 μmol Pi·mg-1 protein·h-1.Our results suggested that membrane vesicles isolated by two-phase method were enriched in sealed and right-side out plasma membrane,and that the optimal pH and temperature of plasma membrane H+-ATPase in P.euphratica callus were 6.5 and 37℃,respectively.更多还原