Binding of [ 3H]forskolin to solubilized preparations of adenylate cyclase

Abstract

The binding of [ 3H]forskolin to proteins solubilized from bovine brain membranes was studied by precipitating proteins with polyethylene glycol and separating [ 3H]forskolin bound to protein from free [ 3H]forskolin by rapid filtration. The K d for [ 3H]forskolin binding to solubilized proteins was 14 nM which was similar to that for [ 3H]forskolin binding sites in membranes from rat brain and human platelets. Forskolin analogs competed for [ 3H]forskolin binding sites with the same rank potency in both brain membranes and in proteins solubilized from brain membranes. [ 3H]forskolin bound to proteins solubilized from membranes with a Bmax of 38 fmol/mg protein which increased to 94 fmol/mg protein when GppNHp was included in the binding assay. In contrast, GppNHp had no effect on [ 3H]forskolin binding to proteins solubilized from membranes preactivated with GppNHp. Solubilized adenylate cyclase from non-preactivated membranes had a basal activity of 130 pmol/mg/min which was increased 7-fold by GppNHp. In contrast, adenylate cyclase from preactivated membranes had a basal activity of 850 pmol/mg/min which was not stimulated by GppNHp or forskolin. Thus, the number of high affinity binding sites for [ 3H]forskolin in solubilized preparations correlated with the activation of adenylate cyclase by GppNHp via the guanine nucleotide binding protein (G s).