Characterization of cholecystokinin receptor sites in guinea-pig cortical membranes using [125I]Bolton Hunter-cholecystokinin octapeptide.
[125I]Bolton Hunter-cholecystokinin octapeptide (BH-CCK8) has been prepared using a modified method and was used to study putative cholecystokinin (CCK) receptor sites in the guinea-pig cerebral cortex. Specific binding of [125I]BH-CCK8, defined as the difference in binding in the absence and presence of 10(-6) M CCK8, was 70% of total binding. In saturation experiments, the apparent dissociation constant (Kd) was 1 nM and total binding capacity was 28 fmol/mg of protein. In association experiments, conducted at 30 degrees C, binding of [125I]BH-CCIK8 reached equilibrium in approximately 150 min. Binding was stable for 4 hr and was reversed by the addition of unlabeled CCK8-sulfated. Dissociation of bound ligand was biphasic and the apparent T1/2 was 45 min. Analyses of kinetic experiments yielded an association rate constant of 0.58 X 10(8) min-1 M-1 and a dissociation rate constant for the slower component of 0.012 min-1. Dithiothreitol increased and N-ethylmaleimide decreased specific binding of [125I]BH-CCK8, indicating that CCK receptor sites involve sulfhydryl groups. In competition experiments, the potency of CCK4 was enhanced 50-fold with addition of protease inhibitors. The rank order of CCK-related peptides was CCK8-sulfated greater than or equal to Gastrin 17 greater than or equal to CCK33 greater than CCK4 greater than or equal to CCK8-desulfated. Proglumide, a proposed CCK antagonist in the periphery and brain, was inactive at 10(-3) M. The specificity of [125I]BH-CCK8 binding sites are similar to that reported for [125I]BH-CCK33.
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D000818:Animals; D002540:Cerebral Cortex / Q000378:metabolism*; D006168:Guinea Pigs; D006801:Humans; D007457:Iodine Radioisotopes; D007700:Kinetics; D009928:Organ Specificity; D011480:Protease Inhibitors / Q000494:pharmacology; D011869:Radioligand Assay; D051381:Rats; D011956:Receptors, Cell Surface / Q000032:analysis*; D011949:Receptors, Cholecystokinin; D012844:Sincalide / Q000031:analogs & derivatives* / Q000138:chemical synthesis; D013045:Species Specificity; D013388:Succinimides / Q000138:chemical synthesis; D013439:Sulfhydryl Reagents / Q000494:pharmacology
C044758:Bolton Hunter-cholecystokinin octapeptide; D007457:Iodine Radioisotopes; D011480:Protease Inhibitors; D011956:Receptors, Cell Surface; D011949:Receptors, Cholecystokinin; D013388:Succinimides; D013439:Sulfhydryl Reagents; D012844:Sincalide