Smigel M D
J Biol Chem. 1986 Feb 5;261(4):1976-82.
The catalytic moiety of hormone-sensitive adenylate cyclase has been purified from bovine brain. It is isolated largely without its guanine nucleotide-binding regulatory protein, Gs, by affinity chromatography on 7-O-hemisuccinyldeacetylforskolin-agarose. It appears to be a single polypeptide which migrates on sodium dodecyl sulfate-polyacrylamide gels with an apparent Mr of approximately 120,000. When subjected to electrophoresis on gradient (5-10%) sodium dodecyl sulfate-polyacrylamide gels, it displays a larger apparent Mr of 150,000. The adenylate cyclase activity of the preparation can be stimulated by the addition of Gs, forskolin, or calcium-calmodulin. The preparation has been reconstituted with purified beta-adrenergic receptors and Gs to form a hormone-stimulated adenylate cyclase system (May, D., Ross, E.M., Gilman, A.G., and Smigel, M.D. (1985) J. Biol. Chem. 260, 15829-15833). In contrast to its stimulation by Gs, inhibition by the alpha subunits of Gi and Go, G proteins known to be coupled to inhibitory receptors (Sternweis, P., and Florio, V. (1985) J. Biol. Chem. 260, 3477-3483), is not seen. Preparations of adenylate cyclase show varying degrees of inhibition by added G protein beta . gamma subunit. This inhibition can be explained as reflecting a variable, small (under 5%) contamination of the preparation by Gs alpha which would be deactivated by complexing with the added beta . gamma subunit.
激素敏感型腺苷酸环化酶的催化部分已从牛脑中纯化出来。通过在7 - O - 半琥珀酰去乙酰佛司可林 - 琼脂糖上进行亲和层析，在很大程度上分离得到了该催化部分，而未得到其鸟嘌呤核苷酸结合调节蛋白Gs。它似乎是一种单一的多肽，在十二烷基硫酸钠 - 聚丙烯酰胺凝胶上迁移时，表观分子量约为120,000。当在梯度（5 - 10%）十二烷基硫酸钠 - 聚丙烯酰胺凝胶上进行电泳时，它显示出更大的表观分子量150,000。该制剂的腺苷酸环化酶活性可通过添加Gs、佛司可林或钙调蛋白来刺激。该制剂已与纯化的β - 肾上腺素能受体和Gs重组，形成了一种激素刺激的腺苷酸环化酶系统（梅，D.，罗斯，E.M.，吉尔曼，A.G.，和斯米格，M.D.（1985年）《生物化学杂志》260，15829 - 15833）。与Gs对其的刺激作用相反，未观察到已知与抑制性受体偶联的G蛋白Gi和Go的α亚基对其的抑制作用（斯特恩韦斯，P.，和弗洛里奥，V.（1985年）《生物化学杂志》260，3477 - 3483）。腺苷酸环化酶制剂对添加的G蛋白βγ亚基表现出不同程度的抑制作用。这种抑制作用可以解释为反映了该制剂被Gsα可变的、少量（低于5%）污染，而Gsα会因与添加的βγ亚基复合而失活。
