Structure of membrane-associated and solubilized uterine adenylate cyclase. Evidence against activation through dissociable subunit interactions in the lipid bilayer.
Adenylate cyclase was extracted from the rat uterus with Lubrol PX in a form which remained soluble following centrifugation for 60 min at 100,000g. The soluble enzyme was stimulated by both Mn+2 and by guanyl-5'-yl-imidodiphosphate (Gpp(NH)p), indicating that both the catalytic subunit (C) and the guanyl nucleotide-binding coupling factor (N) had been extracted. Catalytic activity was bound by a GTP-affinity resin only under conditions which resulted in irreversible activation of the native (particulate) form of the enzyme and could be eluted under acidic conditions shown to reverse the activated state. The S020,w of the soluble enzyme in both its activated and unactivated state was determined by linear sucrose gradient centrifugation. Activation by prolonged treatment with Gpp(NH)p did not alter the S020,w of the enzyme whether treatment was carried out before or after solubilization. The chaotrope LiBr (0.4 M) reduced the S020,w of the soluble enzyme but its smaller size was still not altered by activation with Gpp(NH)p. These results indicate that most adenylate cyclase activity in uterine membranes exists as a preformed complex between the catalytic subunit and the coupling factor: NC. The existence of this complex explains some of the temperature-dependent properties previously described for this form of the enzyme and suggests that dissociable interactions between the subunits do not play a role in the activation of C by guanyl nucleotides.
D000262:Adenylyl Cyclases / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D002462:Cell Membrane / Q000201:enzymology; D002846:Chromatography, Affinity; D003600:Cytosol / Q000201:enzymology; D003902:Detergents; D004789:Enzyme Activation; D005260:Female; D007700:Kinetics; D008051:Lipid Bilayers; D046911:Macromolecular Substances; D000077423:Polidocanol; D011092:Polyethylene Glycols; D051381:Rats; D011919:Rats, Inbred Strains; D012995:Solubility; D014599:Uterus / Q000201:enzymology*
D003902:Detergents; D008051:Lipid Bilayers; D046911:Macromolecular Substances; D000077423:Polidocanol; D011092:Polyethylene Glycols; D000262:Adenylyl Cyclases