一种改进的纯化和鉴定牛脑皮层G_s蛋白的方法

A Modified Method for Purification and Iden-tification of G_s from Bovine Brain Cortex.

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Ｓｅｐｈａｒｏｓｅ６Ｂ柱将两者分开．将含Ｇ＿ｓ高活力的级分用庚胺－Ｓｅｐｈａｒｏｓｅ４Ｂ柱进一步分离，即可获得高活力的Ｇ＿ｓ，ＳＤＳ－ＰＡＧＥ显示为分子量４５０００和３６０００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．

oluble proteins mainly containing G_s (stimu-latory GTP-binding protein) and adenylate cyclase (AC) from cell membranes of bovinebrain cortex were extracted with 1% sodiumcholate and 15% saturated ammonium sulfate.Separation of G_s and AC was carried out bySepharose 6B gel filtration. Purifiled G_s can beobtained by passing the fractions containing G_sfrom Sepharose 6B column through a hepty-laminc Sepharose 4B hydrophobic column.The purity of G_s was identified by its highlystimulated activity to AC and SDS PAGEwhich showed two bands of 45kD and 36kD.The procedure described above is characterizedhy siniplicity. rapidity. repeatability and highyield. At the same time, AC. a by-productwhich was not contaminated by G_s. can beused for assay of G_s activity after reconstitut-ing it into asolectin vesicls. This method ofassaying G_s activity has been proved to be sim-ple. reliable and sensitive.