Physical and immunological characterization of a guanine nucleotide-binding protein purified from bovine cerebral cortex.
Physical and immunological characterization of a guanine nucleotide-binding protein purified from bovine cerebral cortex.
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发表时间：
1985-09
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通讯作者：
R. Huff;J. Axton;E. Neer
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作者：
R. Huff;J. Axton;E. Neer
百日咳毒素的ADP-核糖基化已被用来鉴定Ni的α亚基，它是一种鸟嘌呤核苷酸结合蛋白，介导激素和GTP对腺苷环化酶的抑制。从作为百日咳毒素ADP核糖化底物的牛脑皮质中分离纯化了两种蛋白质，一种是41 kDa的蛋白质(α41)，另一种是39 kDa的蛋白质(α39)。41 kDa的蛋白质与从其他组织中纯化的镍的亚基非常相似，而39 kDa的蛋白质的功能尚不清楚(Neer，E.J.，Lok，J.M.和Wolf，L.G.(1984)J.Biol)。化学。259年，14222-14229；斯特恩维斯，P.C.和罗比肖，J.D.(1984年)J.Biol。化学。259、13806-13813)。我们现在证明，从牛脑中纯化的阿尔法39蛋白是一种相对亲水的蛋白，它与疏水的β-伽马成分有关。该络合物可被5‘-(3-O-硫代)三磷酸鸟苷解离。α39组分与鸟苷5‘-(3-O-硫代)三磷酸结合，Kd值为27 nM。我们已经开发出针对阿尔法39和贝塔的多克隆抗体。在免疫印迹试验中，阿尔法39的抗体与阿尔法41的交叉反应很弱，这表明这两种蛋白质之间存在一定的同源性，但这使得阿尔法39不太可能来自阿尔法41。用这些抗体进行定量，发现膜蛋白中α39为0.5%，β为0.7%。虽然抗体与许多不同物种的α39和β蛋白发生交叉反应，但中枢神经系统组织总是比周围器官的膜具有更强的免疫反应性。当它与阿尔法39或阿尔法41相关联时，抗贝塔抗体识别贝塔亚单位，并能免疫沉淀这两种阿尔法。贝塔伽马三聚体。依赖鸟嘌呤核苷酸的α39的解离。β-伽马三聚体表明，该复合体可能通过释放游离的β-伽马单位来抑制腺苷环化酶。然而，α39的功能可能不是专门调节腺苷环化酶，而是可能包括将激素受体偶联到其他效应器。针对阿尔法39和贝塔的抗体将是确定阿尔法39和贝塔在激素反应细胞中的功能的有用工具。
ADP-ribosylation by pertussis toxin has been used to identify the alpha subunit of Ni, the guanine nucleotide-binding protein which mediates hormone and GTP inhibition of adenylate cyclase. Two proteins have been purified from bovine cerebral cortex which are substrates for ADP-ribosylation by pertussis toxin, a 41-kDa protein (alpha 41) and a 39-kDa protein (alpha 39). The 41-kDa protein is very similar to the subunit of Ni purified from other tissues while the function of the 39-kDa protein is unknown (Neer, E. J., Lok, J. M., and Wolf, L. G. (1984) J. Biol. Chem. 259, 14222-14229; Sternweis, P. C., and Robishaw, J. D. (1984) J. Biol. Chem. 259, 13806-13813). We now show that the purified alpha 39 protein from bovine brain is a relatively hydrophilic protein which associates with a hydrophobic beta gamma component. The complex can be dissociated by guanosine 5'-(3-O-thio)triphosphate. The alpha 39 component binds guanosine 5'-(3-O-thio)triphosphate with a KD of 27 nM. We have developed polyclonal antibodies to alpha 39 and beta. The antibodies to alpha 39 cross-react weakly with alpha 41 in an immunoblot assay indicating some homology between the two proteins but making it unlikely that alpha 39 is derived from alpha 41. Using the antibodies for quantitation we found that alpha 39 is 0.5% and beta is 0.7% of membrane proteins. While the antibodies cross-react with alpha 39 and beta proteins in many different species, central nervous system tissues always have more immunoreactivity than membranes from peripheral organs. Anti-beta antibody recognizes the beta subunit when it is associated with alpha 39 or alpha 41 and can immunoprecipitate both alpha . beta gamma trimers. The guanine nucleotide-dependent dissociation of the alpha 39 . beta gamma trimer suggests that the complex could inhibit adenylate cyclase by liberating free beta gamma units. The function of alpha 39 may not, however, be exclusively to regulate adenylate cyclase but may include coupling hormone receptors to other effectors. Antibodies specific for alpha 39 and beta will be useful tools in determining the functions of alpha 39 and beta in hormone-responsive cells.