Binding of [3H]forskolin to solubilized preparations of adenylate cyclase.
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 Kd 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 (GS).
Nelson
C A
CA
Division of Biochemistry and Biophysics, Food and Drug Administration, Bethesda, MD 20892.
Seamon
K B
KB
D000262:Adenylyl Cyclases / Q000378:metabolism*; D000818:Animals; D001667:Binding, Competitive; D001921:Brain / Q000201:enzymology*; D002417:Cattle; D002462:Cell Membrane / Q000201:enzymology; D005576:Colforsin / Q000031:analogs & derivatives / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology
D005576:Colforsin; D006165:Guanylyl Imidodiphosphate; D000262:Adenylyl Cyclases