Abstract
Activation and regulation of hormone-sensitive adenylate cyclase was studied with forskolin. The models studied are the adenylate cyclase complex in rat erythrocyte membrane, which has minimal sensitivity to hormone regulation and the cyclase complex in the liver membrane, an enzyme complex sensitive to a variety of hormones.Forskolin elicited a significant amount of activity in rat erythrocyte membrane. The enzyme responded typically to epinephrine and GTP. GTP-sensitive and GTP-insensitive states of adenylate cyclase were observed. Hormones (epinephrine or glucagon) facilitate GTP activation and unmask the GTP sensitivity.Preparation of adenylate cyclase of rat liver membrane was improved. The enzyme has low basal and high stimulated activity. Rat liver adenylate cyclase, solubilized with Lubrol PX, has enhanced GTP sensitivity. The unmasking of GTP sensitivity by either stimulatory hormone or Lubrol suggests the presence of factor(s) that regulate the action of GTP and further influence adenylate cyclase already activated by forskolin. The unmasking effect by detergent treatment indicates that the hormone-receptor coupling reaction is not required. A graded activation of adenylate cyclase by forskolin in response to varying concentrations of forskolin, GTP and epinephrine or glucagon was demonstrated. Dose response curves of forskolin in the presence of fixed amount of glucagon or epinephrine, showed a decrease in the EC(,50) of forskolin.Forskolin acts as a stabilizing agent during purification of catalytic component (C). The inactive cyclase was not reactivated by forskolin. C and stimulatory GTP binding protein (N(,s)) from the same tissue can be resolved and functionally isolated on Sephacryl S-300 column. C can be stimulated by forskolin in an assay system but not by GTP. GTP sensitivity in C activation is reconstituted by combining both C and N(,s) fractions. The (alpha)(,s) and (alpha)(,i) subunits of N(,s) and N(,i), were detected using NAD ADP-ribosylation catalysed by cholera toxin and pertussis toxin, respectively.A hypothetical model derived partly from the above, views the forskolin-bound and activated cyclase complex as being further subject to regulation by GTP and N(,s) mediated events. The hypothesis is presented graphically.