Binding of [3H]forskolin to rat brain membranes

K. B. Seamon, Richard Vaillancourt, M. Edwards, J. W. Daly

Research output: Contribution to journalArticle

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Abstract

[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 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 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 languageEnglish (US)
Pages (from-to)5081-5085
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume81
Issue number16 I
StatePublished - 1984
Externally publishedYes

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Colforsin
Membranes
Brain
Binding Sites
Adenylyl Cyclases
Magnesium Chloride
Centrifugation
Proteins
Guanine Nucleotides
Edetic Acid
Spermatozoa
Catalytic Domain
Carrier Proteins

ASJC Scopus subject areas

  • General

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Binding of [3H]forskolin to rat brain membranes. / Seamon, K. B.; Vaillancourt, Richard; Edwards, M.; Daly, J. W.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 81, No. 16 I, 1984, p. 5081-5085.

Research output: Contribution to journalArticle

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