Purification of the catalyst of adenylate cyclase.
The catalytic moiety of hormone-sensitive adenylate cyclase has been purified from bovine brain. It is isolated largely without its guanine nucleotide-binding regulatory protein, Gs, by affinity chromatography on 7-O-hemisuccinyldeacetylforskolin-agarose. It appears to be a single polypeptide which migrates on sodium dodecyl sulfate-polyacrylamide gels with an apparent Mr of approximately 120,000. When subjected to electrophoresis on gradient (5-10%) sodium dodecyl sulfate-polyacrylamide gels, it displays a larger apparent Mr of 150,000. The adenylate cyclase activity of the preparation can be stimulated by the addition of Gs, forskolin, or calcium-calmodulin. The preparation has been reconstituted with purified beta-adrenergic receptors and Gs to form a hormone-stimulated adenylate cyclase system (May, D., Ross, E.M., Gilman, A.G., and Smigel, M.D. (1985) J. Biol. Chem. 260, 15829-15833). In contrast to its stimulation by Gs, inhibition by the alpha subunits of Gi and Go, G proteins known to be coupled to inhibitory receptors (Sternweis, P., and Florio, V. (1985) J. Biol. Chem. 260, 3477-3483), is not seen. Preparations of adenylate cyclase show varying degrees of inhibition by added G protein beta . gamma subunit. This inhibition can be explained as reflecting a variable, small (under 5%) contamination of the preparation by Gs alpha which would be deactivated by complexing with the added beta . gamma subunit.
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D000262:Adenylyl Cyclases / Q000302:isolation & purification* / Q000378:metabolism; D000818:Animals; D002147:Calmodulin / Q000494:pharmacology; D002417:Cattle; D002846:Chromatography, Affinity; D002847:Chromatography, Agarose; D005576:Colforsin / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D019204:GTP-Binding Proteins / Q000378:metabolism; D037102:Lectins / Q000378:metabolism; D008565:Membrane Proteins / Q000302:isolation & purification* / Q000378:metabolism; D014909:Wheat Germ Agglutinins
D002147:Calmodulin; D037102:Lectins; D008565:Membrane Proteins; D014909:Wheat Germ Agglutinins; D005576:Colforsin; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases