Morris D, McHugh-Sutkowski E, Moos M, Simonds W F, Spiegel A M, Seamon K B
Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland 20892.
Biochemistry. 1990 Sep 25;29(38):9079-84. doi: 10.1021/bi00490a027.
An antibody (RM) raised against the carboxyl-terminal decapeptide of the alpha subunit of the stimulatory guanine nucleotide regulatory protein (Gs alpha) has been used to study the interaction of Gs alpha with bovine brain adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1]. RM antibody immunoprecipitated about 60% of the solubilized adenylate cyclase preactivated with either GTP-gamma-S or AlF4-. In contrast, RM antibody immunoprecipitated about 5% of the adenylate cyclase not preactivated (control) and 15% of the adenylate cyclase pretreated with forskolin. Adenylate cyclase solubilized from control membranes or GTP-gamma-S preactivated membranes was partially purified by using forskolin-agarose affinity chromatography. The amount of Gs alpha protein in the partially purified preparations was determined by immunoblotting with RM antibody. There was 3-fold more Gs alpha detected in partially purified adenylate cyclase from preactivated membranes than in the partially purified adenylate cyclase from control membranes. Partially purified adenylate cyclase from preactivated membranes was immunoprecipitated with RM antibody and the amount of adenylate cyclase activity immunoprecipitated (65% of total) corresponded to the amount of Gs alpha protein immunoprecipitated. Only 15% of the partially purified adenylate cyclase from control membranes was immunoprecipitated. The presence of other G proteins in the partially purified preparations of adenylate cyclase was investigated by using specific antisera that detect Go alpha, Gi alpha, and G beta. The G beta protein was the only subunit detected in the partially purified preparations of adenylate cyclase and the amount of G beta was about the same in adenylate cyclase from preactivated membranes and from control membranes.(ABSTRACT TRUNCATED AT 250 WORDS)
一种针对刺激性鸟嘌呤核苷酸调节蛋白（Gsα）α亚基羧基末端十肽产生的抗体（RM），已被用于研究Gsα与牛脑腺苷酸环化酶[ATP焦磷酸裂解酶（环化），EC 4.6.1.1]之间的相互作用。RM抗体免疫沉淀了约60%用GTP-γ-S或AlF4-预激活的可溶性腺苷酸环化酶。相比之下，RM抗体免疫沉淀了约5%未预激活的（对照）腺苷酸环化酶和15%用福斯可林预处理的腺苷酸环化酶。从对照膜或GTP-γ-S预激活膜中溶解的腺苷酸环化酶，通过使用福斯可林-琼脂糖亲和层析进行部分纯化。用RM抗体通过免疫印迹法测定部分纯化制剂中Gsα蛋白的量。在预激活膜的部分纯化腺苷酸环化酶中检测到的Gsα比对照膜的部分纯化腺苷酸环化酶中多3倍。用RM抗体免疫沉淀预激活膜的部分纯化腺苷酸环化酶，免疫沉淀的腺苷酸环化酶活性量（占总量的65%）与免疫沉淀的Gsα蛋白量相对应。对照膜的部分纯化腺苷酸环化酶只有15%被免疫沉淀。通过使用检测Goα、Giα和Gβ的特异性抗血清，研究了腺苷酸环化酶部分纯化制剂中其他G蛋白的存在情况。Gβ蛋白是在腺苷酸环化酶部分纯化制剂中检测到的唯一亚基，并且预激活膜和对照膜的腺苷酸环化酶中Gβ的量大致相同。（摘要截短于250字）
