【摘要】 用1％胆酸钠和20％饱和度的硫酸铵抽提牛脑皮层细胞膜得到含G蛋白和腺苷酸环化酶（AC）的制剂，通过Sepharose6B柱将两者分开，再将含G蛋白的级分用庚胺-Sepharose4B疏水柱、羟基磷灰石柱将其它亚型的G蛋白（主要是Gs和Go）从抑制型G蛋白（Gi）中除去，获得纯化的高活力的Gi，其GTP结合活力为17．6nmol／mg，比细胞膜Gi活力提高50倍；并具有较高的产率，从1g膜蛋白中可获得0．66mg的Gi，同时可获得无G蛋白污染的AC和少量的Gs蛋白．SDS-PAGE显示分子量为41000和36000的两条蛋白带，证实是Gi的α基和β亚基．进一步用重建脂酶体的方法检测Gi对AC的抑制作用，结果显示Gi对AC活力的抑制达40％左右，表明CAMP信息跨膜转导通路中Gi与AC之间具有较好偶联功能．更多还原

【Abstract】 Inhibitory GTP-binding protein(Gi)was exrtacted from the crude membrane of bovine brain by 1% sodium cholate and 20% saturated ammonium sulfate. Purified Gi was obtained by sequential column chromatography on Sepharose 6B,Heptylarnine-Sepharose 4B and hydroxyapatite,and adenylate cyclase (AC )was obtained from the first column of the procedure. The purified G, was identified by its ability to bind specifically guanine nucleotides with higher affinity and SDS-PAGE which showed two bands of 41kD and 36kD. The activity of Gi was assayed by detecting AC activity after incorporating AC and Gi into isolectin liposomes. The result showed that the activity of AC was inhibited by Gi about 40%. This indicates that the coupling function between Gi and AC is acheived. The method of the purifying and assaying Gi has been proved to besimple,repeatible and reliable.更多还原