Evidence for interdependent action of glucagon and nucleotides on the hepatic adenylate cyclase system

Abstract

The kinetic characteristics of glucagon action and binding as functions of hormone and nucleotide concentrations were investigated in the hepatic adenylate cyclase system. Either App(NH)p (5 adenylylimidodiphosphate), 0.1 mM, or adenosine triphosphate, 2 mM, was used as a substrate. The formation of adenosine 3':5' monophosphate (cyclic AMP) in response to glucagon exhibits lag times as long as 4 min in the presence of about 0.1 nM glucagon. The lag times diminish as the hormone or substrate concentrations increase. Addition of guanosine triphosphate (GTP) (0.1 mM) abolishes the lags at all concentrations of glucagon tested in the presence of 0.1 mM App(NH)p and causes a decrease in the concentration of glucagon required for half maximal activation of the enzyme. In the presence of GTP, the apparent K(m) for glucagon action ranges from 0.2 to 0.5 nM glucagon; in its absence, the K(m) ranges from 2 to 7 nM. Pretreatment of the enzyme system with glucagon for 15 min prior to addition of substrate also abolishes the lags seen at low concentrations of the hormone. When the pretreated enzyme system is incubated with limiting concentrations of substrate (0.1 mM App(NH)p) in the absence of free hormone, the velocity of enzyme activity is dependent upon GTP. Stimulatory effects are observed with 30 nM GTP; GTP exerts maximal effects at 1 μm. The lag times observed in onset of glucagon activation appear to reflect a change in state of the enzyme induced by glucagon binding to its receptor. The time dependency is changed simply by increasing the concentration of glucagon; GTP acts subsequent to or attendant upon this change in state induced by glucagon. Glucagon binding exhibits Hill coefficients of about 1.5, suggesting cooperative interactions between two or more subunits of the system. GTP, in addition to its effects on glucagon action, stimulates the rate of dissociation of glucagon from its receptor and, as a consequence, causes a decrease in the steady state levels of bound glucagon. The release of hormone seems not to be causally related to enzyme activation since the rates of dissociation are increased by addition of 0.1 mM GTP without concomitant increases in enzyme velocity. A hyperbolic relationship is observed between the levels of receptors occupied by glucagon and adenylate cyclase activity generated in the presence of GTP. Complete activation of the enzyme requires full occupation of the specific binding sites in hepatic plasma membranes (about 3 pmoles per mg of membrane protein). This finding shows that all of the specific binding sites for glucagon in these membranes represent receptors functionally and structurally linked to the adenylate cyclase system. The finding that glucagon and GTP activate adenylate cyclase by a concerted or interdependent mechanism is discussed in relation to the characteristics of the adenylate cyclase system as an allosteric regulatory enzyme system.