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 (NIH), Bethesda, MD (United States)
- OSTI ID:
- 6027231
- Journal Information:
- Proc. Natl. Acad. Sci. U.S.A.; (United States), Vol. 81:16
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
59 BASIC BIOLOGICAL SCIENCES
TERPENES
CHEMICAL BONDS
AFFINITY
BRAIN
CALCIUM CHLORIDES
CELL MEMBRANES
CENTRIFUGATION
CYCLASES
ENZYME ACTIVITY
HORMONES
ISOTHERM
MAGNESIUM CHLORIDES
MANGANESE CHLORIDES
MATHEMATICAL MODELS
PROTEINS
RATS
SODIUM CHLORIDES
TRACER TECHNIQUES
TRITIUM COMPOUNDS
ALKALI METAL COMPOUNDS
ALKALINE EARTH METAL COMPOUNDS
ANIMALS
BODY
CALCIUM COMPOUNDS
CALCIUM HALIDES
CELL CONSTITUENTS
CENTRAL NERVOUS SYSTEM
CHLORIDES
CHLORINE COMPOUNDS
ENZYMES
HALIDES
HALOGEN COMPOUNDS
ISOTOPE APPLICATIONS
LABELLED COMPOUNDS
LYASES
MAGNESIUM COMPOUNDS
MAMMALS
MANGANESE COMPOUNDS
MANGANESE HALIDES
MEMBRANES
NERVOUS SYSTEM
ORGANIC COMPOUNDS
ORGANS
RODENTS
SEPARATION PROCESSES
SODIUM COMPOUNDS
TRANSITION ELEMENT COMPOUNDS
VERTEBRATES
560305* - Chemicals Metabolism & Toxicology- Vertebrates- (-1987)
550201 - Biochemistry- Tracer Techniques