A kinetic analysis of activation of smooth muscle adenylate cyclase by forskolin

Forskolin action was studied using uterine smooth muscle adenylate cyclase, an enzyme form that is slowly and irreversibly activated by treatment with nonhydrolyzable GTP analogs. Activation of the particulate smooth muscle enzyme by prolonged treatment with GppNH]p (guanyl-5′-yl imidodiphosphate) at 24 °C followed simple Michaelis-Menten kinetics with respect to the guanine nucleotide. Under these treatment conditions, forskolin increased both the V_max and the K_m for GppNH]p, suggesting diterpene action affected the guanine nucleotide-binding coupling factor. Sensitivity of a detergent-solubilized form of the enzyme to stimulation by both GppNH]p and forskolin was much more labile at 4 °C than was the Mn⁺² sensitivity of the catalytic subunit. In the particulate form, the catalytic subunit was more resistant to the denaturing effects of N-ethylmaleimide than was its sensitivity to stimulation by GppNH]p or forskolin. Forskolin stimulation of the particulate form of the enzyme followed simple Michaelis-Menten kinetics with respect to the concentration of the diterpene. Denaturation of the enzyme by treatment with N-ethylmaleimide lowered the V_max and increased the K_m for forskolin, further suggesting that forskolin had an indirect effect on the activity of the catalytic subunit. These results could be accounted for if the diterpene, like GppNH]p, was bound by the coupling factor.