Northup J K, Smigel M D, Sternweis P C, Gilman A G
J Biol Chem. 1983 Sep 25;258(18):11369-76.
Activation of the stimulatory guanine nucleotide-binding regulatory component (G/F) of adenylate cyclase by guanine nucleotides or by Al3+, Mg2+, and F-stabilizes the protein to thermal denaturation or to inactivation by LiBr, guanidine HCl, or urea. Such activation allows the resolution of the active 45,000-Da alpha subunit from the 35,000-Da beta subunit by a high performance gel filtration procedure. Separation of the active alpha subunit has allowed definitive evaluation of the subunit dissociation model for the activation of G/F. The resolved alpha subunit is sufficient to reconstitute the adenylate cyclase activity of the cyc-S49 cell mutant. The alpha subunit alone is also sufficient to activate a preparation of the catalyst of adenylate cyclase that had been resolved from all other identified components of the enzyme system. The resolved alpha subunit displays hydrodynamic properties characteristic of activated G/F. The alpha subunit contains a high affinity guanine nucleotide-binding site. Activation of G/F by guanine nucleotides or by Al3+ + Mg2+ + F- allows resolution of the activated alpha subunit. Reversal of the activated state of the resolved alpha subunit occurs only slowly. Addition of beta subunit enhances the rate of deactivation. Deactivation of the activated alpha subunit by the beta subunit changes the S20,w for G/F activity from 2.0 to 4.0 (in Lubrol), consistent with a formation of the alpha X beta heterodimer. These data, taken in aggregate, constitute proof for the proposed mechanism of activation of G/F by non-hydrolyzable analogs of GTP and by Al3+, Mg2+, and F-. They are analogous to data obtained for transducin, the GTP-binding regulatory protein from vertebrate rod outer segment discs, and for the putative inhibitory guanine nucleotide-binding regulatory component of adenylate cyclase (the substrate for islet-activating protein). The model provides several powerful tests for study of mechanisms of hormonal regulation of adenylate cyclase in membranes.
鸟嘌呤核苷酸或Al3+、Mg2+和F-对腺苷酸环化酶刺激性鸟嘌呤核苷酸结合调节成分（G/F）的激活作用，可使该蛋白对热变性或被LiBr、盐酸胍或尿素失活产生稳定作用。这种激活作用使得通过高效凝胶过滤程序能够从35,000道尔顿的β亚基中分离出活性45,000道尔顿的α亚基。活性α亚基的分离使得能够对G/F激活的亚基解离模型进行确切评估。分离得到的α亚基足以重建cyc-S49细胞突变体的腺苷酸环化酶活性。单独的α亚基也足以激活从酶系统的所有其他已鉴定成分中分离出来的腺苷酸环化酶催化剂制剂。分离得到的α亚基表现出激活的G/F所特有的流体动力学性质。α亚基含有一个高亲和力的鸟嘌呤核苷酸结合位点。鸟嘌呤核苷酸或Al3+ + Mg2+ + F-对G/F的激活作用使得能够分离出激活的α亚基。分离得到的α亚基激活状态的逆转仅缓慢发生。添加β亚基可提高失活速率。β亚基使激活的α亚基失活会使G/F活性的S20,w从2.0变为4.0（在十二烷基硫醇中），这与αXβ异二聚体的形成一致。综合这些数据，构成了对GTP不可水解类似物以及Al3+、Mg2+和F-激活G/F的 proposed机制的证据。它们类似于从脊椎动物视杆细胞外段盘的转导素（一种GTP结合调节蛋白）以及腺苷酸环化酶假定的抑制性鸟嘌呤核苷酸结合调节成分（胰岛激活蛋白的底物）获得的数据。该模型为研究膜中腺苷酸环化酶的激素调节机制提供了几个有力的测试方法。
