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Rapid kinetics of myo-inositol trisphosphate binding and dissociation in cerebellar microsomes
Z Hannaert-Merah1, J F Coquil, L Combettes
1Unité de Recherche Associée 1290 (Centre National de la Recherche Scientifique, Département de Biologie Cellulaire et Moléculaire Commissariat à l'Energie Atomique), Centre d'Etudes de Saclay, Gif-sur-Yvette, France.
Abstract:
Using sheep cerebellum microsomes adsorbed on a filter, we measured the kinetics of [3H]inositol 1,4,5-trisphosphate (InsP3) binding and dissociation on the subsecond time scale during rapid perfusion of the filter with [3H]InsP3-containing or InsP3-free media. At 20 degrees C and pH 7.1, in a cytosol-like medium containing MgCl2, the half-time for InsP3 dissociation was as short as 125 ms. The receptor behaved as a simple target for binding of its ligand, with the rate constant for InsP3 binding increasing linearly with InsP3 concentration. Various modulators of InsP3 binding (KCl, NaCl, pH, Mg2+, and Ca2+) were found to affect the receptor's apparent affinity for InsP3 mainly by altering the rate constant for [3H]InsP3 dissociation. ATP (but not InsP3) also accelerated [3H]InsP3 dissociation. In contrast to these modulators, luminal Ca2+ was found to have no effect on the amount of microsome-bound [3H]InsP3.