Synthesis of 2',3'-O-(2,4,6-trinitrocyclohexadienylidine)guanosine 5'-triphosphate and a study of its inhibitory properties with adenylate cyclase.
A fluorescent GTP analog 2',3'-O-(2,4,6-trinitrocyclohexadienylidine) guanosine 5'-triphosphate (TNP-GTP) has been prepared and some of its physical properties characterized. TNP-GTP was found to be a potent inhibitor of chick embryo heart adenylate cyclase as activated by guanyl 5'-(beta,gamma-imido)triphosphate (GppNHp), F-, and forskolin with Ki values in the 8-15 microM range. It also appeared to inhibit substantially basal adenylate cyclase in this system. TNP-GTP demonstrated an effective competition with [3H]GppNHp, binding to membranes equivalently to GppNHp and about three times better than GTP. 8-Azidoguanosine 5'-triphosphate (8N3GTP) mimics GTP activation of chick embryo heart adenylate cyclase and [gamma-32P]8N3GTP is effectively photoincorporated into a 42,000- to 44,000-Mr doublet when proteins are separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. TNP-GTP effectively prevents this photoincorporation, as does GTP, at concentrations that agree with their respective apparent inhibition and activation binding constants. The data suggest that TNP-GTP could prove to be a valuable tool for studying the mechanisms of GTP regulation of adenylate cyclase and other GTP-regulated systems.
D000067956:Adenylyl Cyclase Inhibitors; D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D001386:Azides / Q000378:metabolism; D001667:Binding, Competitive; D055598:Chemical Phenomena; D002621:Chemistry; D002642:Chick Embryo; D005576:Colforsin / Q000037:antagonists & inhibitors; D004789:Enzyme Activation / Q000187:drug effects; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000378:metabolism / Q000494:pharmacology; D006165:Guanylyl Imidodiphosphate / Q000037:antagonists & inhibitors / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D009206:Myocardium / Q000201:enzymology; D010777:Photochemistry; D012969:Sodium Fluoride / Q000037:antagonists & inhibitors; D013053:Spectrophotometry
C021961:8-azidoguanosine triphosphate; D000067956:Adenylyl Cyclase Inhibitors; D001386:Azides; D005576:Colforsin; D006165:Guanylyl Imidodiphosphate; D006160:Guanosine Triphosphate; D012969:Sodium Fluoride; C045093:2',3'-O-(2,4,6-trinitrocyclohexadienylidene)guanosine 5'-triphosphate; D000469:Alkaline Phosphatase