Figure 3.

InsP₃ binding reveals three binding sites. (A) Saturation-binding isotherm of InsP₃ binding to cerebellar reticular vesicles. InsP₃ binding was conducted in the absence of Ca with InsP₃ concentrations varying between 0.4 nM and 30 μM. Inset shows the Scatchard analysis. Data were fit assuming the presence of three binding sites for InsP₃. Four different vesicle preparations were used; one experiment is displayed. In the absence of Ca, the K _d's for the vesicle preparations are 0.47 ± 0.19 nM, 54 ± 5 nM, and 10.2 ± 3.2 μM; x̄ ± SEM, n = 4. (B) Saturation-binding isotherm of InsP₃ binding to the type 1 InsP₃ receptor purified by heparin-agarose chromatography. Inset shows the Scatchard analysis. Similar results were obtained with a receptor purified from cerebellar microsomes by immunoprecipitation using a type 1-specific antibody (C-19). The purity of the receptor, as assessed from InsP₃ binding, was 94% (InsP₃ binding was 3,612 pmol/ mg for the 50-nM site, compared with a theoretical maximum binding of 3,846 pmol/mg for a molecular weight of 260 kD). In this panel, the abscissa is expressed as picomoles per microliter of purified InsP₃ receptor. K _d's for this preparation are 0.5 nM, 29 nM, and 6.8 μM. For comparison, one preparation of InsP₃ receptor purified by immunoprecipitation generated K _d's of 0.1 nM, 35 nM, and 12 μM. (C) Scatchard analysis of InsP₃ binding in the absence of Ca (•) and 10 μM free Ca (○). Increasing the free Ca results in a decrease in the apparent affinity of InsP₃ binding from 35 nM in the absence of Ca to 91 nM in the presence of 10 μM Ca without altering binding to the 1-nM or 10-μM sites. The Ca-induced decrease in InsP₃ affinity of the 50-nM site was completely reversed upon addition of EGTA.