In vitro comparisons of hepatic adenylate cyclase from normal and hypophysectomized rat liver plasma membrane.

Abstract

The adenylate cyclase activity in both hypophysectomized (hypox) and normal rat liver plasma membrane (RLM) has been characterized and compared in an attempt to decipher the molecular differences of the adenylate cyclase in these two membrane systems. Basal and glucagon- and fluoride-stimulated adenylate cyclase activities were severalfold greater in hypox RLM than in normal RLM. The elevation in adenylate cyclase activity in hypox RLM occurred within 36-48 h posthypophysectomy. The time course of stimulation with both glucagon and guanyl-5'-imidodiphosphate of the adenylate cyclase in both hypox and normal RLM was characterized by increased activity. The kinetics of activation of adenylate cyclase indicated similar Km values for hypox and normal RLM for both glucagon- and fluoride-stimulated activity, whereas the maximum velocity (Vmax) was 2- to 3-fold greater for the hypox RLM compared to the normal RLM. The adenylate cyclase activities in both normal and hypox RLM exhibited similar responses to increases in Mg++ or Mn++ concentrations. Manganese concentrations greater than 10 mM returned the glucagon-stimulated activity to the basal level in both membrane systems. The fluoride-stimulated activity in both hypox and normal RLM was about 40% of the maximum activity even at 20 mM Mn++. The activity of the hypox RLM was greater than the normal RLM at all Mn++ concentrations. Calcium inhibited adenylate cyclase activity in both normal and hypox RLM in a similar manner. A rapid decrease in fluoride-stimulated activity in these two membrane systems was observed above 3-5 mM Ca++. Maximum inhibition of glucagon-stimulated cyclase activity in normal RLM occurred at about 0.5 mM Ca++, while more than 3 mM Ca++ was required to decrease the hypox RLM glucagon-stimulated activity to levels observed in normal RLM. EGTA enhanced the activity of adenylate cyclase in both normal and hypox RLM. The enhancement was characterized by a 2.7-fold increase with a peak at 0.15 mM EGTA in hypox RLM, and a 2-fold increase without a well defined peak between 0.05-0.3 mM EGTA in normal RLM. The pH dependencies of glucagon- and fluoride-stimulated adenylate cyclase activities in normal and hypox RLM were somewhat different. The pH values for maximum activity for normal RLM and hypox RLM were 7.5 and 8.0, respectively. The results of this study suggested that basic differences exist between adenylate cyclase in hypox and normal RLM and that the increased adenylate cyclase activity in hypox RLM resides in the catalytic unit of the cyclase.