Binding of (/sup 3/H)Forskolin to rat brain membranes
(12-/sup 3/H)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 MgCl/sub 2/ by using the centrifugation assay is described best by a two-site model: K/sub d1/ = 15 nM, B/sub max/sub 1// (maximal binding) = 270 fmol/mg of protein; K/sub d2/ = 1.1 ..mu..M; B/sub max/sub 2// = 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/sub d/ = 26 nM, B/sub 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 (/sup 3/H)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 MgCl/sub 2/ or MnCl/sub 2/ 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/sub d/. There is no effect of CaCl/sub 2/ (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. 23 references, 5 figures, 2 tables.
- Research Organization:
- National Institutes of Health, Bethesda, MD
- OSTI ID:
- 6027231
- Journal Information:
- Proc. Natl. Acad. Sci. U.S.A.; (United States), Journal Name: Proc. Natl. Acad. Sci. U.S.A.; (United States) Vol. 81:16; ISSN PNASA
- Country of Publication:
- United States
- Language:
- English
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560305* -- Chemicals Metabolism & Toxicology-- Vertebrates-- (-1987)
59 BASIC BIOLOGICAL SCIENCES
63 RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTANT EFFECTS ON LIVING ORGS. AND BIOL. MAT.
AFFINITY
ALKALI METAL COMPOUNDS
ALKALINE EARTH METAL COMPOUNDS
ANIMALS
BODY
BRAIN
CALCIUM CHLORIDES
CALCIUM COMPOUNDS
CALCIUM HALIDES
CELL CONSTITUENTS
CELL MEMBRANES
CENTRAL NERVOUS SYSTEM
CENTRIFUGATION
CHEMICAL BONDS
CHLORIDES
CHLORINE COMPOUNDS
CYCLASES
ENZYME ACTIVITY
ENZYMES
HALIDES
HALOGEN COMPOUNDS
HORMONES
ISOTHERM
ISOTOPE APPLICATIONS
LABELLED COMPOUNDS
LYASES
MAGNESIUM CHLORIDES
MAGNESIUM COMPOUNDS
MAMMALS
MANGANESE CHLORIDES
MANGANESE COMPOUNDS
MANGANESE HALIDES
MATHEMATICAL MODELS
MEMBRANES
NERVOUS SYSTEM
ORGANIC COMPOUNDS
ORGANS
PROTEINS
RATS
RODENTS
SEPARATION PROCESSES
SODIUM CHLORIDES
SODIUM COMPOUNDS
TERPENES
TRACER TECHNIQUES
TRANSITION ELEMENT COMPOUNDS
TRITIUM COMPOUNDS
VERTEBRATES