Reconstitution of cholera toxin-activated adenylate cyclase.
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.
D000067956:Adenylyl Cyclase Inhibitors; D000262:Adenylyl Cyclases / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000187:drug effects; D002460:Cell Line; D002772:Cholera Toxin / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects; D005033:Ethylmaleimide / Q000494:pharmacology; D005459:Fluorides / Q000494:pharmacology; D006160:Guanosine Triphosphate / Q000378:metabolism; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology; D046911:Macromolecular Substances; D009154:Mutation
D000067956:Adenylyl Cyclase Inhibitors; D046911:Macromolecular Substances; D006165:Guanylyl Imidodiphosphate; D006160:Guanosine Triphosphate; D002772:Cholera Toxin; D000262:Adenylyl Cyclases; D005033:Ethylmaleimide; D005459:Fluorides