Effects of sulfhydryl reagents on [3H] inositol trisphosphate binding to dog cerebellar membranes.
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 (Bmax = 44 pmol/mg protein, Kd = 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-chloromercuricphenyl 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 microM, 70 microM, 1 mM, and 0.1 mM, respectively. The inhibition was specific since the presence of 10 microM 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.
Yang
C M
CM
Department of Physiology, Lyon Laboratory, University of Minnesota, Minneapolis 55455.
Lee
H C
HC
D000818:Animals; D001665:Binding Sites; D015220:Calcium Channels; D002531:Cerebellum / Q000378:metabolism*; D004285:Dogs; D066298:In Vitro Techniques; D015544:Inositol 1,4,5-Trisphosphate; D053496:Inositol 1,4,5-Trisphosphate Receptors; D007295:Inositol Phosphates / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000032:analysis*; D018160:Receptors, Cytoplasmic and Nuclear; D013403:Sugar Phosphates / Q000378:metabolism*; D013439:Sulfhydryl Reagents / Q000494:pharmacology*
D015220:Calcium Channels; D053496:Inositol 1,4,5-Trisphosphate Receptors; D007295:Inositol Phosphates; D011956:Receptors, Cell Surface; D018160:Receptors, Cytoplasmic and Nuclear; D013403:Sugar Phosphates; D013439:Sulfhydryl Reagents; D015544:Inositol 1,4,5-Trisphosphate