摘要
谷胱甘肽转移酶(EC 2,5,1,18 Glutathione S-transferases简称GSTs)是一组具有多种生理功能的蛋白质。我们通过105,000×g超速离心,s—已基—谷胱甘肽—Sepharose-6B亲和层析柱和DEAE52纤维柱或CM52纤维柱将人肝粗匀浆纯化为电泳纯的GSTs同工酶。经系和层析柱后GSTs比活比粗匀浆上清液提高54倍,回收率近60％。通过DE52柱将人肝GSTs分离为7个同工酶组分,分别称为c_(DE),A_1,A_2,A_3,A_4,A_5和A_6,经等电聚焦电泳和SDS-pAGE电泳鉴定,其等电点依次为8.60,7.05,6.70,6:60,6.55,6.45和6.4。经CM52柱后得到5个不同的同工酶组分,分别定名为A_(CM),c_1,c_2,c_3和c_4等电点各自为 6.30,7.00,8.50,8.55和8.60。阳离子同工酶(即c_(DE),C_1,C_2,C_3和C_4)的分子量在23,500—24,000道尔顿,阴离子同工酶(A_(CM),A_1-A_6)约为25,000道尔顿。并将亲和层析柱后样品,阳离子同工酶C_(DE)和阴离子同工酶A_(CM)作为抗原,得到兔抗人肝GSTs相应同工酶的抗血清,其抗血清效价经免疫双扩散法测定分别为1:96,1:64,1:16。并对人肝GSTs进行氨基酸组份的测定。
The glutathions S-transferases (EC 2.5.1.18. GSTs) were purified approximately 54 fold to electrophoretic homogenity from 105,000 X g of human liver by affinity chromatography on S-hexylglutathione linked Sepharose-6B.This purification procedure resulted in 60% yield of the original GSTs activity toward 1-chloro -2, 4-dinitrobezene. Further purification by CM-cellulose and DEAE-cellulose column cbromatography resolved the GSTs preparation into five cationic isozymes designated as CDE, C-1, C-2, C-3. and C-4 and seven anionic isozymes designated as ACM, A-1, A-2, A-3, A-4, A-5 and A-6 in the order of their elution off the ion-exchange colomn. SDS-PAGE electrophoretic data on subunit composition revealed that cationic enzymes were composed of Ya subunit with an identical Mr of 24,000,wheras a predominant subunit with Mr of 25,000 was observed in all anionic isozymes peaks. Isoelectric points of cationic isozymes were 8.50-8.60 and anionic isozymes 6.30-7.05. Ouchterlony double-diffusion expeliments showed a cross reaction of our antiserum raised against Chinese liver GgTs with America liver, but not with the rat liver GSTs.
关键词
人肝
GSTS
亲和层析
Glutathione S-transferases (GSTs) Human liver Affinity chomatography
