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.
Department of Pharmacology, University of Connecticut Health Center, Farmington 06032.
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O'Rourke
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Feinstein
D001120:Arginine; D001665:Binding Sites; D001667:Binding, Competitive; D001792:Blood Platelets / Q000378:metabolism*; D015220:Calcium Channels; D002462:Cell Membrane / Q000378:metabolism; D016264:Dextran Sulfate; D003911:Dextrans / Q000494:pharmacology*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D015544:Inositol 1,4,5-Trisphosphate / Q000097:blood*; D053496:Inositol 1,4,5-Trisphosphate Receptors; D007295:Inositol Phosphates / Q000097:blood*; D007700:Kinetics; D011956:Receptors, Cell Surface / Q000187:drug effects / Q000378:metabolism*; D018160:Receptors, Cytoplasmic and Nuclear; D013464:Sulfuric Acids / Q000494:pharmacology*
D015220:Calcium Channels; D003911:Dextrans; C506926:ITPR1 protein, human; D053496:Inositol 1,4,5-Trisphosphate Receptors; D007295:Inositol Phosphates; D011956:Receptors, Cell Surface; D018160:Receptors, Cytoplasmic and Nuclear; D013464:Sulfuric Acids; D015544:Inositol 1,4,5-Trisphosphate; D016264:Dextran Sulfate; D001120:Arginine