The glucagon-sensitive adenyl cyclase system in plasma membranes of rat liver. V. An obligatory role of guanylnucleotides in glucagon action.

A method is described for the enzymatic synthesis of 5’-adenylyl imidodiphosphate labeled with a2P at the a! position (AMP-PNP-cx-~~P), an analogue of ATP containing nitrogen substituted for oxygen between the terminal phosphates. The nucleotide is only slowly hydrolyzed during incubation with rat liver plasma membranes and is a substrate for adenyl cyclase in these membranes. In the presence of 0.2 mM AMP-PNP, glucagon and fluoride ion stimulate adenyl cyclase activity; linear rates are maintained for at least 10 min of incubation at 30”. GTP enhanced the initial rate of basal and glucagon-stimulated adenyl cyclase activity. Reduction in concentration of Mg++ in the assay medium or incubation of liver membranes for 5 min at 30” prior to addition of glucagon results in loss of response of adenyl cyclase to glucagon and in reduction in the effects of GTP on basal activity. Under these conditions GTP, GDP, or GMP-PCP are required for glucagon stimulation of the enzyme even though the specific binding sites for glucagon are saturated with hormone; as little as 10 nM GTP or GDP is required. UTP and CTP exert smaller effects than the guanyl nucleotides and act only at concentrations higher than 0.1 mm. The guanyl nucleotides inhibited the response of adenyl cyclase to fluoride ion (10 mu) over the same concentration range over which they stimulate the response of the enzyme to glucagon. This action of the nucleotides is observed in plasma membranes treated with phospholipase A under conditions that result in loss of glucagon binding and of hormonal response. It is concluded that guanyl nucleotides play a specific and obligatory role in the activation of adenyl cyclase by glucagon. The nucleotides bind at sites, distinct from the glucagon binding sites, that appear to regulate both the response of adenyl cyclase to glucagon, and, possibly by a related mechanism, the actions of fluoride ion on this system.