PMID: 6539776Jun 25, 1984Paper

Evidence for a single forskolin-binding site in rat adipocyte membrane. Studies of [14,15-3H]dihydroforskolin binding and adenylate cyclase activation.

The Journal of Biological Chemistry
R J Ho, Q H Shi

Abstract

[14,15-3H] Dihydroforskolin has been used as a tracer in the study of forskolin binding to adipocyte plasma membrane and the subsequent activation of adenylate cyclase (EC 4.6.1.1) of this membrane. The specific binding of radioactivity to the membrane was rapid, temperature-dependent, saturable, and readily reversible. The equilibrium dissociation reaction constant (KD) for the binding was 13 microM, with a maximum binding (Bmax) of 61 pmol of forskolin per mg of membrane protein. The Hill coefficient was 1.0. The bound [14,15-3H] dihydroforskolin was displaced by forskolin with rate constants of 0.07 X 10(6) M-1 min-1 and 1.2 min-1 for the association and dissociation reactions, respectively (30 degrees C). The equilibrium dissociation constant (KD) was approximately the same as the concentration that produced half-maximum activation (EC50) of the adenylate cyclase of rat adipocyte plasma membrane. There was a linear correlation between forskolin binding and adenylate cyclase activation. The results are consistent with the concept of a single class of binding site which binds forskolin. [14,15-3H] Dihydroforskolin appears to be a potentially useful tracer in the study of the mechanism of activation of the catalytic unit of ad...Continue Reading

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