Identification and purification from bovine brain of a guanine-nucleotide-binding protein distinct from Gs, Gi and Go.
Identification and purification from bovine brain of a guanine-nucleotide-binding protein distinct from Gs, Gi and Go.
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从牛脑中鉴定和纯化不同于 Gs、Gi 和 Go 的鸟嘌呤核苷酸结合蛋白。
DOI：
10.1042/bj2460431
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发表时间：
1987
期刊：
影响因子：
--
通讯作者：
Harden,TK
中科院分区：
文献类型：
--
作者：
Waldo,GL;Evans,T;Fraser,ED;Northup,JK;Martin,MW;Harden,TK
用DEAE-Sephacel、Ultrogel AcA-34、庚胺-Sepharose和Sephadex G-150连续层析从牛脑膜胆酸盐提取物中纯化了一种鸟嘌呤核苷酸结合蛋白（G蛋白）。鸟苷5′-[γ-[35 S]硫代]三磷酸（GTP[35 S]）结合活性与25，000 Da肽和35，000 - 36，000 Da蛋白质双联体共纯化。百日咳毒素和霍乱毒素都不能催化与GTP[35 S]结合活性相关的蛋白质的ADP-核糖基化。用8-叠氮基[γ-32 P]GTP对纯化的蛋白质进行光亲和标记，表明GTP结合位点位于25，000 Da蛋白质上。35，000 - 36，000 Da蛋白质双联体与其他GTP结合蛋白的β亚基在电泳上无法区分，36，000 Da蛋白质可被寡聚体GT的抗血清识别。纯化的蛋白质特异性结合17.2 nmol GTP[35 S]/mg蛋白质。放射性配体的结合位点的Kd约为1.5kD。15 nM。在SDS/聚丙烯酰胺凝胶电泳过程中，脑GTP结合蛋白与从人胎盘纯化的称为Gp的GTP结合蛋白共迁移[Evans，Brown，Fraser & Northup（1986）J.Biol.Chem.261，7052-7059]，并与抗胎盘蛋白的抗血清交叉反应，但不与抗脑Go的抗血清交叉反应。在金黄色葡萄球菌V8蛋白酶存在下，脑和胎盘GTP结合蛋白的SDS/聚丙烯酰胺凝胶电泳产生相同的肽图。
A guanine-nucleotide-binding protein (G-protein) was purified from cholate extracts of bovine brain membranes by sequential DEAE-Sephacel, Ultrogel AcA-34, heptylamine-Sepharose and Sephadex G-150 chromatography. Guanosine 5′-[gamma-[35S]thio]triphosphate (GTP[35S])-binding activity copurified with a 25,000 Da peptide and a 35,000-36,000 Da protein doublet. Neither pertussis toxin nor cholera toxin catalysed the ADP-ribosylation of a protein associated with the GTP[35S]-binding activity. Photoaffinity labelling of the purified protein with 8-azido[gamma-32P]GTP indicated that the GTP-binding site resides on the 25,000 Da protein. The 35,000-36,000 Da protein doublet was electrophoretically indistinguishable from the beta-subunits of other GTP-binding proteins, and the 36,000 Da protein was recognized by antiserum to oligomeric Gt. The purified protein specifically bound 17.2 nmol of GTP[35S]/mg of protein. The Kd of the binding site for radioligand was approx. 15 nM. The brain GTP-binding protein co-migrated during SDS/polyacrylamide-gel electrophoresis with a GTP-binding protein, named Gp, purified from human placenta [Evans, Brown, Fraser & Northup (1986) J. Biol. Chem. 261, 7052-7059], and cross-reacted with antiserum raised against the placental protein, but not with antiserum raised to brain Go. SDS/polyacrylamide-gel electrophoresis of the brain and placental GTP-binding proteins in the presence of Staphylococcus aureus V8 protease yielded identical peptide maps.