Binding of 5' guanylyl imidodiphosphate to turkey erythrocyte membranes and effects on β adrenergic activated adenylate cyclase

5' Guanylyl imidodiphosphate (Gpp(NH)p), an analog of GTP, augments isoproterenol stimulated adenylate cyclase causing greater activation of the enzyme than with 7 mM sodium fluoride. The effect occurs with a Km for Gpp(NH)p of 10^-7M. A similar Km was observed for the slight stimulation of the enzyme by the nucleotide in the absence of added hormone. The greatly enhanced stimulation of adenylate cyclase activity by isoproterenol in the presence of Gpp(NH)p is blocked completely by propranolol. The latter does not, however, abolish the stimulation of basal activity produced by the nucleotide. The Km for isoproterenol activation of adenylate cyclase is decreased one order of magnitude by Gpp(NH)p. Gpp(NH)p binds to the cell membranes with a Km equivalent to that found for effects on adenylate cyclase activity. Propranolol does not inhibit binding of the nucleotide, but unlabeled Gpp(NH)p and other nucleotides competitively displace labeled Gpp(NH)p from its binding sites with apparent affinities comparable to the order of potencies for augmenting catecholamine stimulated adenylate cyclase. Displacement of Gpp(NH)p from the binding site by GTP or ITP (GTP was 100 times as effective as ITP) decreases enzyme activity activated by isoproterenol in the presence of Gpp(NH)p. Thus, a high affinity purine nucleotide site is involved in regulating the catalytic function of the adenylate cyclase complex. The apparent affinity of the site for ATP is much lower than for GTP or Gpp(NH)p. This finding indicates that, although ATP is clearly the substrate for the enzyme, it is GTP and not ATP that interacts at the regulatory site (identified as a Gpp(NH)p binding site).