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. 1991 Jun 15;276(Pt 3):621–630. doi: 10.1042/bj2760621

Evidence for regulation of human platelet adenylate cyclase by phosphorylation. Inhibition by ATP and guanosine 5'-[beta-thio]diphosphate occur by distinct mechanisms.

I A Wadman 1, R W Farndale 1, B R Martin 1
PMCID: PMC1151050  PMID: 1905923

Abstract

1. Incubation of human platelet membranes with guanosine 5'-[beta gamma-imido]triphosphate (p[NH]ppG) causes a time-dependent increase in the activation of adenylate cyclase due to Gs (the stimulatory GTP-binding protein). Forskolin enhances adenylate cyclase activity but does not interfere with the process of activation. The activation follows first-order kinetics in both the presence and the absence of the assay components. 2. ATP in the presence or the absence of an ATP-regenerating system of phosphocreatine and creatine kinase inhibits activation. 3. Hydrolysis of ATP to ADP does not lead to receptor-mediated inhibition of adenylate cyclase acting via Gi (the inhibitory GTP-binding protein). The ADP analogue adenosine 5'-[beta-thio]diphosphate (ADP[S]) does not inhibit the activation process. 4. Phosphocreatine alone inhibits adenylate cyclase activation at concentrations above 1 mM. 5. Inhibition by phosphocreatine is not due to the chelation of free Mg2+ ions. 6. Inhibition by ATP and the other assay components occurs throughout the activation process, decreasing both the rate of activation and the maximum activity obtained. 7. Maximal activation of adenylate cyclase after prolonged incubation with p[NH]ppG slowly reverses in the presence of the assay components. 8. A 10-fold excess of the GDP analogue guanosine 5'-[beta-thio]diphosphate (GDP[S]) over p[NH]ppG inhibits the activation process completely, at all stages of the time course. 9. Preincubations in the presence and absence of ATP, cyclic AMP, phosphocreatine and creatine kinase show equal sensitivity to increasing GDP[S] concentration. These data show that the inhibition observed in the presence of ATP is not due to endogenous or contaminating guanine nucleotides, and suggest that phosphoryl transfer may regulate adenylate cyclase activity.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

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