Arad H, Rosenbusch J P, Levitzki A
Proc Natl Acad Sci U S A. 1984 Nov;81(21):6579-83. doi: 10.1073/pnas.81.21.6579.
Turkey erythrocyte membranes were solubilized in the mild detergent octylpenta(oxyethylene) [CH3(CH2)7-(OCH2CH2)5OH], which possesses a high critical micelle concentration (approximately equal to 6 mM) and forms small, dynamic micelles. Both the native enzyme Ns(GDP) X C and the p[NH]ppG-preactivated species N's X p[NH]ppG X C' were found to possess the same molecular mass of 215,000 +/- 17,000 daltons. Both enzyme species migrate as a tight complex between Ns and C on both gel permeation columns and on DEAE-Sephacel columns in detergent. The two functional units, Ns and C, remain associated even in dilute detergent solutions and throughout a 300- to 400-fold purification in octylpoly(oxyethylene). These results strongly support the view that Ns and C do not come apart during the process of enzyme activation by the beta-adrenergic receptor. Furthermore, these results strongly support our previous assertion that the beta-adrenergic receptor activation of adenylate cyclase is by a simple "collision coupling" between the receptor and NsC. These results are not compatible with shuttle mechanisms that postulate that Ns physically migrates from the receptor R to the catalytic unit C and back during the activation cycle, as suggested by Citri and Schramm [Citri, Y. & Schramm, M. (1980) Nature (London) 287, 297-300] and by De Lean et al. [De Lean, A., Stadel, J. M. & Lefkowitz, R. J. (1980) J. Biol. Chem. 255, 5108-5117].
土耳其红细胞膜在温和去污剂辛基聚（氧乙烯）[CH3(CH2)7-(OCH2CH2)5OH]中溶解，该去污剂具有高临界胶束浓度（约等于6 mM），并形成小的动态胶束。发现天然酶Ns(GDP)X C和p[NH]ppG预激活物种N's X p[NH]ppG X C'具有相同的分子量，为215,000 +/- 17,000道尔顿。这两种酶在去污剂中的凝胶渗透柱和DEAE-葡聚糖凝胶柱上均以Ns和C之间的紧密复合物形式迁移。即使在稀释的去污剂溶液中以及在辛基聚（氧乙烯）中进行300至400倍纯化的过程中，两个功能单元Ns和C仍保持结合。这些结果有力地支持了这样的观点，即Ns和C在β-肾上腺素能受体激活酶的过程中不会分离。此外，这些结果有力地支持了我们先前的论断，即β-肾上腺素能受体对腺苷酸环化酶的激活是通过受体与NsC之间简单的“碰撞偶联”实现的。这些结果与穿梭机制不相符，穿梭机制假定Ns在激活周期中从受体R物理迁移到催化单元C并返回，如Citri和Schramm [Citri, Y. & Schramm, M. (1980) Nature (London) 287, 297-300]以及De Lean等人[De Lean, A., Stadel, J. M. & Lefkowitz, R. J. (1980) J. Biol. Chem. 255, 5108-5117]所提出的那样。
