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′-(β,γ-imido)triphosphate (GppNHp), F-, and forskolin with Ki values in the 8-15 μm 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 [γ-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.
ASJC Scopus subject areas
- Molecular Biology