The irreversible activation of adenylate-cyclase by 5'guanylylimidodiphosphate, a phosphoramidate analog of 5'GTP, has been examined in toad (Bufus marinus) plasma membranes using the technique of preincubating the membranes with the nucleotide under various controlled conditions followed by washing and subsequent assay of enzyme activity. Activation of adenylate cyclase by Gpp(NH)p, but not GTP, is essentially permanent and persists following extensive washing, prolonged incubation at 30 degrees C in the absence of the nucleotide, and after dissolution of the membranes with Lubrol PX. (-)-Isoproterenol increases the activation observed with maximal concentrations of Gpp(NH)p from eight- to 10-fold (in the absence of hormone) to 50- to 100-fold; final activities as high as 10-15 nmoles of cyclic AMP per min per mg protein are achieved. The activated state obtained with isoproterenol and Gpp(NH)p is also permanent and is not inhibited by propranolol. The synergism between Gpp(NH)p and hormone requires the simultaneous presence of these compounds, and the time-dependent enhancement of activation with (-)-isoproterenol may be interrupted by addition of propranolol. The stimulation is slow, and may proceed for as long as 45 min at 30 degrees C in the presence of maximal concentrations of Gpp(NH)p and (-)-isoproterenol. Very little activation occurs at 0 degrees C. The time course of activation at 30 degrees C exhibits an accelerating phase lasting from 5 to 30 min when Gpp(NH)p is added directly during assay of cyclase activity or when the membranes are preincubated for various times and washed prior to assay for a fixed time. The lag period occurs in the presence and absence of (-)-isoproterenol, although the rate of increase in velocity is greater with hormone. The length of the accelerating phase decreases with increasing concentrations of Gpp(NH)p, although it is still evident with maximal levels of Gpp(NH)p and hormone. However, prewarming the membranes at 30 degrees C for 10 min in the absence of Gpp(NH)p or (-)-isoproterenol results in an immediate onset of linear activation at a rate which is achieved in untreated membranes only after about 10 min. The events occurring during prewarming at 30 degrees C are readily reversible since chilling the warmed membranes to 0 degrees C results in a time course of activation identical to that of membranes maintained at 0 degrees C until addition of Gpp(NH)p. Activation is proportional to the concentration of Gpp(NH)p within the range of 10(-8) to 10(-4) mM. The apparent affinity for Gpp(NH)p increases with increasing time of incubation. The primary effect of increasing the concentration of Gpp(NH)p is to decrease the time required to obtain a maximal rate of activation. The possible relevance of these findings to the mechanism of action of Gpp(NH)p, adenylate cyclase and hormones is discussed within the context of current views of biological membranes which recognize the lateral mobility of membrane molecules.
Adenylate Cyclase • Animals • Bufo marinus • Cell Membrane • Dose-Response Relationship, Drug • Drug Synergism • Enzyme Activation • Erythrocytes • Guanosine Triphosphate • Isoproterenol • Kinetics • Temperature • analogs & derivatives* • blood* • enzymology • enzymology* • pharmacology • ultrastructure