Characterization of [3H](+/-)L-364,718 binding to solubilized cholecystokinin (CCK) receptors of rat pancreas.

The binding of 3HL-364,718 (3S(-)-N-(2,3-dihydro-1-methyl-2-oxo-5-phenyl-1H-1,4-benzodiazepine-3-yl )-1H-indole-2-carboxamide), an extremely potent nonpeptide cholecystokinin (CCK) receptor antagonist, to digitonin-solubilized CCK receptors from rat pancreas was characterized. 3HL-364,718 binding to digitonin-solubilized receptors was assayed using polyethylene glycol precipitation followed by rapid filtration to separate free and bound 3HL-364,718. Specific 3HL-364,718 binding to solubilized receptors was dependent on the digitonin and receptor concentration and, under optimal conditions, represented greater than 90% of the total binding. Scatchard analysis indicated a single class of binding sites with a Kd of 0.53 nM and a Bmax of 3.1 pmol/mg protein. Specific 3HL-364,718 binding to solubilized CCK receptors was inhibited by both CCK receptor agonists and antagonists in a stereospecific manner. After solubilization, the affinities of various antagonists to displace specific 3HL-364,718 binding were similar to those obtained with membrane-bound receptors; however, the affinities of CCK agonists were reduced 10-100 times. Collectively, the data presented indicate that 3HL-364,718 represents a new antagonist ligand which has apparent advantages over the agonist ligand [125I]CCK in assaying digitonin-solubilized receptors. Gel filtration of the digitonin-solubilized CCK receptors followed by 3HL-364,718 binding determinations revealed an estimated molecular weight of 400,000 daltons.