一种改进的纯化和鉴定牛脑皮层Gs蛋白的方法

用1%胆酸钠和15%饱和硫酸铵相结合的方法,从牛脑皮层细胞膜中抽提得到主要含激活型G-蛋白(G_s)和腺苷酸环化酶(AC)两种蛋白组分的制剂,然后通过Sepharose 6B柱将两者分开.将含G_s高活力的级分用庚胺-Sepharose 4B柱进一步分离,即可获得高活力的G_s,SDS-PAGE显示为分子量45 000和36 000的两条蛋白带.该法具有简便、快速、重复性好、产率高等优点,且可同时获得无G_s污染的AC.用无G_s污染的AC脂酶体测定G_s活力亦简便、可靠、灵敏度高.

A Modified Method for Purification and Identification of Gs from Bovine Brain Cortex

Soluble proteins mainly containing G_s (stimulatory GTP-binding protein) and adenylate cyclase (AC) from cell membranes of bovine brain cortex were extracted with 1% sodium cholate and 15% saturated ammonium sulfate. Separation of G_s and AC was carried out by Sepharose 6B gel filtration. Purifiled G_s can be obtained by passing the fractions containing G_s from Sepharose 6B column through a heptylaminc Sepharose 4B hydrophobic column. The purity of G_s was identified by its highly stimulated activity to AC and SDS PAGE which showed two bands of 45kD and 36kD. The procedure described above is characterized by simplicity, rapidity, repeatability and high yield.At the same time,AC,a by-product which was not contaminated by G_s, can be used for assay of G_s, activity after reconstituting it into asolectin vesicls. This method of assaying Gs activity has been proved to be simple, reliable and sensitive.