Johnson G L, Kaslow H R, Bourne H R
Proc Natl Acad Sci U S A. 1978 Jul;75(7):3113-7. doi: 10.1073/pnas.75.7.3113.
Reconstitution of adenylate cyclase activity responsive to stimulation by guanylyl-5'imidodiphosphate or NaF may be achieved by mixing dilute Lubrol 12A9-solubilized extracts of wild-type S49 membranes with membranes of an adenylate cyclase-deficient variant. Experiments using N-ethylmaleimide to inactivate components of the adenylate cyclase system indicate that distinct components from both wild-type detergent extracts and adenylate cyclase-deficient membranes are essential for reconstitution. These results and conclusions confirm those of E. M. Ross and A. G. Gilman [J. Biol. Chem. (1977) 252, 6966-6969]. Detergent extracts of cholera toxin-treated wild-type membranes yield a reconstituted adenylate cyclase as responsive to GTP as to guanylyl-5'-imidodiphosphate whereas, in the absence of cholera toxin treatment, GTP has little or no effect. Cholera toxin-treated adenylate cyclase-deficient membranes and Lubrol 12A9 extracts from them, however, fail to yield a reconstituted adenylate cyclase that responds to GTP with an increase in cyclase activity. Because treatment of the adenylate cyclase-deficient variants with cholera toxin is without effect on the reconstituted cyclase, we propose that the cholera toxin substrate is absent or altered in the adenylate cyclase-deficient phenotype.
通过将野生型S49细胞膜的稀释Lubrol 12A9增溶提取物与腺苷酸环化酶缺陷变体的膜混合，可以实现对鸟苷酰-5'-亚氨基二磷酸或NaF刺激有反应的腺苷酸环化酶活性的重建。使用N-乙基马来酰亚胺使腺苷酸环化酶系统的成分失活的实验表明，来自野生型去污剂提取物和腺苷酸环化酶缺陷膜的不同成分对于重建至关重要。这些结果和结论证实了E.M.罗斯和A.G.吉尔曼的研究结果[《生物化学杂志》（1977年）252卷，6966 - 6969页]。霍乱毒素处理的野生型膜的去污剂提取物产生的重建腺苷酸环化酶对GTP的反应与对鸟苷酰-5'-亚氨基二磷酸的反应一样灵敏，而在没有霍乱毒素处理的情况下，GTP几乎没有影响。然而，霍乱毒素处理的腺苷酸环化酶缺陷膜及其Lubrol 12A9提取物未能产生对GTP有反应且环化酶活性增加的重建腺苷酸环化酶。由于用霍乱毒素处理腺苷酸环化酶缺陷变体对重建的环化酶没有影响，我们提出在腺苷酸环化酶缺陷表型中霍乱毒素底物不存在或发生了改变。
