Kinetic identification of a two-state glucagon receptor system in isolated hepatocytes. Interconversion of homogeneous receptors.

A detailed kinetic study was performed to investigate the interaction of glucagon with receptors on freshly isolated hepatocytes. Competition binding assay results fit a mathematical expression for a single site noncooperative model of binding. Glucagon was shown to bind with first-order kinetics at six-hormone concentrations (0.02-0.50 nM) at 0 and 37 degrees C. The observed pseudo-first-order rate constants are directly proportional to the hormone concentration at 0 degree C, but display a downward deviation from linearity at 37 degrees C. Dissociation of glucagon exhibited biexponential character at 37 degrees C which was not seen at 0 degree C. The biphasic dissociation at 37 degrees C was resolved into rapid (t1/2 = 1.9 min) and slow (t1/2 = 27.7 min) components. The distribution of the total bound hormone between the rapidly and slowly dissociating complexes was not dependent upon the extent of receptor occupancy. The absolute quantity of rapidly dissociating hormone-receptor complexes was constant at all times examined; however, the fraction of slowly dissociating hormone-receptor complexes was found to increase with increasing incubation time. The results indicate that a homogeneous population of hepatic receptors undergoes a time-dependent, temperature-dependent conversion from one state to another in a two-stage sequential manner.