Separation of a guanine nucleotide regulatory unit from the adenylate cyclase complex with GTP affinity chromatography.
We studied the relationship between guanine nucleotide binding proteins and adenylate cyclase activity of solubilized turkey erythrocyte membranes with GTP affinity chromatography. Solubilized adenylate cyclase from untreated membranes or membranes pretreated with GMP only (GMP prep) responded poorly to GTP or Gpp(NH)p but were markedly stimulated by fluoride. The solubilized enzyme from membranes pretreated with isoproterenol+GMP (ISO+GMP prep) did not respond to GTP but was markedly stimulated by Gpp(NH)p. Fluoride-stimulated activity of the ISO+GMP prep was reduced by comparison with the GMP prep but could be increased significantly by addition of GTP. GTP hexane agarose (GTP linked to matrix via morpholine derivative of ribose) failed to interact specifically with either the GMP or ISO+GMP prep. Incubation of ISO+GMP prep with GTP-gamma-agarose (GTP linked to matrix via terminal phosphate) reduced the Gpp(NH)p response and the GTP-dependent fraction of the fluoride response. GTP-gamma-agarose did not reduce the fluoride response of the GMP prep. Following incubation with ISO+GMP prep, GTP-gamma-agarose beads were eluted with buffer containing Gpp(NH)p. The eluate had only slight intrinsic adenylate cyclase activity but was able to increase significantly the activity of GTP-gamma-agarose-treated ISO+GMP prep as well as untreated GMP prep. An eluate from GTP-gamma-agarose beads incubated with GMP prep did not possess activity. Our results suggest that the guanine nucleotide-regulatory unit is reversibly associated with the adenylate cyclase complex of turkey erythrocyte membranes.
D000262:Adenylyl Cyclases / Q000032:analysis* / Q000378:metabolism; D000818:Animals; D002352:Carrier Proteins / Q000302:isolation & purification*; D002846:Chromatography, Affinity / Q000379:methods; D004789:Enzyme Activation / Q000187:drug effects; D004910:Erythrocyte Membrane / Q000201:enzymology*; D004912:Erythrocytes / Q000201:enzymology*; D006150:Guanine Nucleotides / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology; D046911:Macromolecular Substances; D012995:Solubility; D014422:Turkeys
D002352:Carrier Proteins; D006150:Guanine Nucleotides; D046911:Macromolecular Substances; D006165:Guanylyl Imidodiphosphate; D006160:Guanosine Triphosphate; D000262:Adenylyl Cyclases