Adenosine 5′-(β, γ-imino)triphosphate and guanosine 5′-O-(2-thiodiphosphate) do not necessarily provide non-phosphorylating conditions in adenylate cyclase studies

Abstract

Commercial preparations of adenosine 5′-(β, γ-imino)triphosphate (App(NH)p) were found to be contaminated with a GTP-like substance(s) as well as a phosphate donor(s) for GDP. Thus, when these preparations were used as substrate with no purification, GDP was as effective as GTP in promoting PGE 1 stimulation of human platelet adenylate cyclase. With purified App(NH)p as substrate, the effect of PGE 1 with GDP was reduced but still observable, while that with GTP was unaltered. PGE 1 also caused a stimulation in the presence of guanosine 5′-o-(2-thiodiphosphate) (GDPβS) with ATP as substrate. Both of the PGE 1-stimulated activities observed with GDP and its analog were completely lost by the addition of UDP, thereby, inhibiting GTP formation catalized by membrane-associated nucleoside diphosphate kinase. The results demonstrate that the stimulatory effects of PGE 1 observed with GDP and App(NH)p, and with GDPβS and ATP were transphosphorylation dependent and, therefore, the analogs must be used with special caution in adenylate cyclase studies.