Evidence for multiple interconvertible forms of adenylate cyclase detected by forskolin activation.

Activation of luteal adenylate cyclase has been measured as a function of forskolin concentration using 15 mM Mg2+ and 2, 5 and 15 mM Mn2+ as divalent cation. Analysis of these data demonstrates that activation is best described by a mixture of two binding sites which differ in their affinity for forskolin. The apparent dissociation constants of about 0.5 microM and 15 microM showed little change with type or concentration of metal ion. The overall increase in activation over basal activity (delta Vmax) was highest with Mg2+ at 1.17 nmol/min/mg protein, decreased about 10% with 2 mM Mn2+ to 1.02 pmol/min/mg and about 20% with 5 and 15 mM Mn2+ (approximately 0.9 nmol/min/mg). The major effect of increasing Mn2+ ion is to alter the amount of activation attributable to each binding site. At 2 mM Mn2+ 74% of the activation is associated with the low affinity site (Kd approximately 15 microM) compared to 80% with Mg2+. In contrast, only 41% of the activation is associated with the low affinity site using 5 and 15 mM Mn2+. In a separate experiment 5 and 15 mM Mn2+ were found to cause uncoupling of Gpp (NH) p activation. These results suggest that the different affinities for forskolin are related to differences in the interaction between the regulatory and catalytic subunit and indicate that forskolin activation will be a useful tool in studying the regulation of adenylate cyclase.