Effects of Sulfhydryl Reagents on [³H] Inositol Trisphosphate Binding to Dog Cerebellar Membranes

Abstract

Homogenates from dog cerebellum were fractionated using sucrose gradient centrifugation. The [³H]inositol 1, 4, 5-trisphosphate binding and the glucose 6-phosphatase activities were found to co-purify. The binding was saturable and had high affinity (B_max=44 pmol/mg protein, K_d=116 nM). Selective chemical modification was used to examine amino acid residues of the microsomal receptor that might be critical for the binding of inositol trisphophate. Sulfhydryl reagents, p-chloromercuric-phenyl sulfonic acid, eosin 5-maleimide, N-ethyl maleimide and fluorescein 5-maleimide were found to be highly potent inhibitors of the binding with half-maximal inhibition occurring at about 20 µM, 70 µM, 1 mM, and 0.1 mM, respectively. The inhibition was specific since the presence of 10 µM of inositol trisphosphate during the reaction completely protected against the inhibition by these reagents. These results suggest that sulfhydryl group is essential for inositol trisphosphate binding to its receptor.