Stimulation of catecholamine-sensitive adenylate cyclase by 5'-guanylyl-imidodiphosphate.

The GTP analog 5’-guanylyl-imidodiphosphate (Gpp(NH)p) caused marked stimulation of basal and catecholamine (isoproterenol)-sensitive adenylate cyclase from canine myocardium, frog erythrocytes and rat paraovarian fat. The combination of Gpp(NH)p (1OV M) and isoproterenol (10e4 M) produced activation of adenylate cyclase equal to (fat) or significantly greater than (heart, erythrocytes) that stimulated by fluoride ion. The great activity of Gpp(NH)p was not due to its greater resistance to hydrolysis than GTP or to inhibition of ATP hydrolysis, since an ATP regenerating system was employed in the adenylate cyclase assays, and no significant hydrolysis of ATP or GTP occurred. GTP caused stimulation of adenylate cyclase in myocardial membranes, was virtually without effect in frog erythrocytes and resulted in inhibition in adipose membranes. Despite the much greater enzyme activation by Gpp(NH)p than GTP, the affinity of GTP for the nucleotide regulatory sites on the enzyme was greater. GTP potently and competitively antagonized the greater enzyme stimulation by Gpp(NH)p. Adenylate cyclase stimulation by any concentration of Gpp(NH)p was 50% inhibited by a lo-fold lower concentration of GTP. The concentration of Gpp(NH)p necessary for half-maximal stimulation was doubled by 1OV M GTP. Ability to competitively antagonize stimulation by Gpp(NH)p provided a convenient way of assessing the relative affinities of nucleotides for the regulatory sites. These a5nities were GTP > GDP > GMP > ITP with UTP and CTP either very weak or inert. Since GTP and other nucleotides are capable of causing only partial activation of the enzyme, comparison of their affinities for the nucleotide regulatory sites on the basis of ability to stimulate adenylate cyclase may be misleading. This is particularly true in frog erythrocyte membranes where the guanine nucleotides have almost no intrinsic activity although they may have high affinity for the sites. The marked stimulation of adenylate cyclase by Gpp(NH)p appears to be due to an increase in the Vmax of the