Huff R M, Axton J M, Neer E J
J Biol Chem. 1985 Sep 5;260(19):10864-71.
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.
百日咳毒素介导的 ADP-核糖基化已被用于鉴定 Ni 的α亚基，Ni 是一种鸟嘌呤核苷酸结合蛋白，可介导激素和 GTP 对腺苷酸环化酶的抑制作用。从牛脑皮质中纯化出了两种蛋白质，它们是百日咳毒素进行 ADP-核糖基化的底物，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出的 Ni 亚基非常相似，而 39 kDa 蛋白质的功能尚不清楚（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）。我们现在表明，从牛脑中纯化出的α39 蛋白质是一种相对亲水的蛋白质，它与一种疏水的βγ组分结合。该复合物可被鸟苷 5'-(3 - O - 硫代)三磷酸解离。α39 组分以 27 nM 的 KD 值结合鸟苷 5'-(3 - O - 硫代)三磷酸。我们已制备了针对α39 和β的多克隆抗体。在免疫印迹分析中，针对α39 的抗体与α41 有微弱的交叉反应，这表明这两种蛋白质之间存在一些同源性，但不太可能α39 是由α41 衍生而来。使用这些抗体进行定量分析，我们发现α39 占膜蛋白的 0.5%，β占 0.7%。虽然这些抗体在许多不同物种中与α39 和β蛋白都有交叉反应，但中枢神经系统组织的免疫反应性总是比外周器官的膜更强。抗β抗体在β与α39 或α41 结合时能识别β亚基，并且可以免疫沉淀α·βγ三聚体。α39·βγ三聚体的鸟嘌呤核苷酸依赖性解离表明，该复合物可能通过释放游离的βγ亚基来抑制腺苷酸环化酶。然而，α39 的功能可能并不完全是调节腺苷酸环化酶，还可能包括将激素受体与其他效应器偶联。针对α39 和β的特异性抗体将是确定α39 和β在激素反应性细胞中的功能的有用工具。
