title: A second guanyl nucleotide-binding site associated with adenylate cyclase. Distinct nucleotides activate adenylate cyclase and permit ADP-ribosylation by cholera toxin.
abstract: There are two functionally and physically distinct types of guanyl nucleotide site associated with the adenylate cyclase system of pigeon erythrocytes. One is on the well known regulatory protein, N, that mediates the adenylate cyclase response to hormones, guanyl nucleotides and fluoride, and is the substrate for ADP-ribosylation by cholera toxin. We now describe a second site that must be occupied by GTP or an analog of GTP before N can be ADP-ribosylated. We call this second site S. It differs from the site on N in many respects. GTP appears to be rapidly hydrolyzed when it is bound to N but not when bound at S. GTP analogs such as guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) bind stably to both sites but the binding of GTP gamma S to N is more sensitive to EDTA and is more easily prevented by guanosine 5'-O-(2-thiodiphosphate). The nucleotide binding only to S is promoted by the cytosolic protein required by cholera toxin. Isoproterenol decreases GTP gamma S binding to S while indirectly increasing GTP gamma S binding to N. By adjusting the binding conditions, the nucleotides bound functionally to N and S can be varied independently and then the effect of ADP-ribosylation upon the adenylate cyclase activity can be seen to depend on the type of nucleotide bound to N. This activity rises, falls slightly, or remains at zero, if N is occupied by GTP, GTP gamma S, or guanosine 5'-O-(2-thiodiphosphate, respectively.
mesh_terms: D000246:Adenosine Diphosphate Ribose / Q000378:metabolism*; D000262:Adenylyl Cyclases / Q000097:blood*; D000818:Animals; D002772:Cholera Toxin / Q000494:pharmacology*; D010856:Columbidae; D003600:Cytosol / Q000201:enzymology; D004912:Erythrocytes / Q000201:enzymology*; D019204:GTP-Binding Proteins; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D007545:Isoproterenol / Q000494:pharmacology; D007700:Kinetics; D009702:Nucleoside Diphosphate Sugars / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000097:blood*; D013873:Thionucleotides / Q000494:pharmacology
chemical_list: D009702:Nucleoside Diphosphate Sugars; D011956:Receptors, Cell Surface; D013873:Thionucleotides; D000246:Adenosine Diphosphate Ribose; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate; D002772:Cholera Toxin; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases; D007545:Isoproterenol