Molecular complexes involved in the regulation of adenylate cyclase

Selective extraction of the adenylate cyclase regulatory protein (N-protein) from pigeon erythrocyte plasma membranes provided evidence for its cytoskeletal association. Cholate, but not Triton X-100 or digitonin, was effectivie in solubilizing the ADP-ribosylated N-protein. The labeled protein complex or components thereof that were associated with the Triton-insoluble cytoskeleton (shells) could be partly released by 0.1 mM EDTA; 1 M KCl in the presence of Triton X-100 achieved complete solubilization. 5'-Guanylyl imidodiphosphate (p[NH]ppG) and NaF, activators of adenylate cyclase, promoted the release of the regulatory protein from the cytoskeleton but MnCl₂ an 'uncoupler' of the adenylate cyclase system, had the opposite effect. The solubilized, labeled N-protein was able to bind specifically to rat erythrocyte inside-out vesicles in the presence of divalent cations. A proteolytic product of inside-out vesicles inhibited the binding of the N-protein to fresh vesicles. Three molecular species which contained the Mr 45,000 polypeptide component of the N-protein were identified by gel permeation chromatography and by sucrose density gradient velocity sedimentation. p[NH]ppG appeared to convert the two larger molecular complexes to a smaller molecular entity. Such a molecular dissociation might be relevant to the effect of guanyl nucleotides on the activity of adenylate cyclase and on the affinity of hormone receptors.