Inositol 1,4,5-trisphosphate slowly converts its receptor to a state of higher affinity in sheep cerebellum membranes.
Coquil J.F. | Mauger J.P. | Claret M.
Incubation of cerebellar microsomes with D-myo-inositol 1,4,5-trisphosphate (InsP3) (0.01-1 micromolar), at 4 or 20 degrees C in a cytosolic-like medium devoid of Ca2+ and Mg2+, followed by InsP3 removal, induced an increase in InsP3 binding determined with 1 nM [3H]InsP3. At 20 degrees C, and pH 7.1, maximal stimulation (1.5-2.5-fold) was obtained with 1 micromolar InsP3, and the EC50 was 60 +/- 5 nM. Several lines of evidence suggested that the activating site is identical with the InsP3 binding site: (i) activation and binding exhibited the same inositol phosphate specificity; (ii) addition of decavanadate, a competitive inhibitor of [3H]InsP3 binding, to the preincubation mixture, prevented the activating effect of InsP3; (iii) the concentration of InsP3 giving half-maximal activation was close to that giving half-maximal InsP3 binding. The time course of activation was found to be much slower than that of binding. While a t1/2 less than 0.4 s has been measured recently at neutral pH and 20 degrees C for binding of 0.5 nM [3H]InsP3 (Hannaert-Merah, Z., Coquil, J.-F., Combettes, L., Claret, M., Mauger, J.-P., and Champeil, P. (1994) J. Biol. Chem. 269, 29642-29649), a 20-s preincubation with 1 micromolar InsP3 was required to half-maximally stimulate binding. Under the present conditions, the InsP3-induced binding increase was only partially reversible. However, this effect was not blocked by antiproteases suggesting that it did not involve proteolysis. Taking advantage of the marked difference in the kinetics of InsP3 binding and InsP3-dependent activation, we performed binding experiments on a short period (3 s) to determine the effect of InsP3 pretreatment on the binding parameters. The data showed that this treatment increased the affinity of the receptor without changing the number of binding sites (control: KD = 107 nM, Bmax = 28 pmol/mg of protein; after preincubation with 1 micromolar InsP3: KD = 53 nM, Bmax = 32 pmol/mg of protein). The two states of the.
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