The inositol 1,4,5-trisphosphate receptor binding sites of platelet membranes. pH-dependency, inhibition by polymeric sulphates, and the possible presence of arginine at the binding site.
The present study was initiated to characterize the inositol 1,4,5-trisphosphate (InsP3)-binding site in human platelets that is involved in Ca2+ release. InsP3 binding to platelet membranes was measured in two ways; (1) by displacement of labelled InsP3 with unlabelled InsP3, as in previous studies, and (2) directly, using only radioactive InsP3 as ligand, over the concentration range 0.25-100 nM. At physiological pH (7.1) the binding data were best fitted by a model for a single saturable binding site, with KD = 11.8 nM and Bmax. = 1.4 pmol/mg of protein. At alkaline pH values (8.3 and 9.4) binding was best fitted by a two-site model, the second site being of higher affinity (KD = 0.75-1.2 nM) but lower concentration (Bmax. = 0.195-0.6 pmol/mg of protein). All binding of InsP3 was blocked by polymeric sulphates (heparin, dextran sulphate, polyvinyl sulphate) regardless of pH. The specific arginine-modifying reagent p-hydroxyphenylglyoxal irreversibly blocked InsP3 binding, suggesting the presence of arginine at the recognition site for InsP3 binding. NN'-dicyclohexylcarbodi-imide (DCCD) and 1-ethyl-3-(3-dimethylaminopropyl)carbodi-imide (ECCD), which are carboxy-group-specific reagents, blocked Ca2+ release, but not InsP3 binding, indicating the existence of another site that regulates Ca2+ release apart from the active centre for InsP3.