Binding of [3H]forskolin to rat brain membranes.
[12-3H]Forskolin (27 Ci/mmol) has been used to study binding sites in rat brain tissue by using both centrifugation and filtration assays. The binding isotherm measured in the presence of 5 mM MgCl2 by using the centrifugation assay is described best by a two-site model: Kd1 = 15 nM, Bmax1 (maximal binding) = 270 fmol/mg of protein; Kd2 = 1.1 microM; Bmax2 = 4.2 pmol/mg of protein. Only the high-affinity binding sites are detected when the binding is determined by using a filtration assay; Kd = 26 nM, Bmax = 400 fmol/mg of protein. Analogs of forskolin that do not activate adenylate cyclase (EC 4.6.1.1) do not compete effectively for [3H]forskolin binding sites. Analogs of forskolin that are less potent than forskolin in activating adenylate cyclase are also less potent in competing for forskolin binding sites. The presence of 5 mM MgCl2 or MnCl2 was found to enhance binding. In the presence of 1 mM EDTA the amount of high-affinity binding is reduced to 110 fmol/mg of protein with no change in Kd. There is no effect of CaCl2 (20 mM) or NaCl (100 mM) on the binding. No high-affinity binding can be detected in membranes from ram sperm, which contains an adenylate cyclase that is not activated by forskolin. It is proposed that the high-affinity binding sites for forskolin are associated with the activated complex of catalytic subunit and stimulatory guanine nucleotide binding protein.
D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D001667:Binding, Competitive; D001921:Brain / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism*; D002413:Cations, Divalent; D002462:Cell Membrane / Q000378:metabolism; D005576:Colforsin; D004224:Diterpenes / Q000378:metabolism*; D004492:Edetic Acid / Q000494:pharmacology; D007700:Kinetics; D008297:Male; D051381:Rats; D013094:Spermatozoa / Q000378:metabolism; D014316:Tritium
D002352:Carrier Proteins; D002413:Cations, Divalent; D004224:Diterpenes; D014316:Tritium; D005576:Colforsin; D004492:Edetic Acid; D000262:Adenylyl Cyclases