Stimulatory GTP regulatory unit N(S) and the catalytic unit of adenylate cyclase are tightly associated: Mechanistic consequences

Turkey erythrocyte membranes were solubilized in the mild detergent octylpenta(oxyethylene) [CH₃(CH₂)₇-(OCH₂CH₂)₅OH], which possesses a high critical micelle concentration (~6 mM) and forms small, dynamic micelles. Both the native enzyme N(S)(GDP)·C and the p[NH]ppG-preactivated species N'(S)·p[NH]ppG·C' were found to possess the same molecular mass of 215,000 ± 17,000 daltons. Both enzyme species migrate as a tight complex between N(S) and C on both gel permeation columns and on DEAE-Sephacel columns in detergent. The two functional units, N(S) 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 N(S) and C do not come apart during the process of enzyme activation by the β-adrenergic receptor. Furthermore, these results strongly support our previous assertion that the β-adrenergic receptor activation of adenylate cyclase is by a single 'collision coupling' between the receptor and N(S)C. These results are not compatible with shuttle mechanisms that postulate that N(S) 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].