[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: K(d1) = 15 nM, B(max1) (maximal binding) = 270 fmol/mg of protein; K(d2) = 1.1 μM; B(max2) = 4.2 pmol/mg of protein. Only the high-affinity binding sites are detected when the binding is determined by using a filtration assay; K(d) = 26 nM, B(max) = 400 fmol/mg of protein. Analogs of forskolin that do not activate adenylate cyclase (EC 126.96.36.199) 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 K(d). 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 associated with the activated complex of catalytic subunit and stimulatory guanine nucleotide binding protein.
|Original language||English (US)|
|Number of pages||5|
|Journal||Proceedings of the National Academy of Sciences of the United States of America|
|Issue number||16 I|
|State||Published - Jan 1 1984|
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