Solubilization and characterisation of the cholecystokininB binding site from pig cerebral cortex.
Cholecystokinin (CCK) binding sites were solubilized from pig cerebral cortical membranes with digitonin (2%, w/v) in the presence of Na+ (120 mM) and Mg2+ (5 mM). Scatchard plot transformation of equilibrium binding data obtained with 125I-CCK-8S gave an apparent dissociation constant (Kd) of 0.6 nM, comparable to that obtained in membranes in the presence of these cations. Hill coefficients close to unity suggested the presence of a single population of receptor sites. Competitive inhibition studies with pentagastrin, gastrin(1-17)S and the CCKA receptor antagonist L-364,718 indicated that the solubilized receptor sites were of the B-type (CCKB), with the same pharmacological profile as that observed in membranes. Optimal specific binding of 125I-CCK-8S to membrane-bound and solubilized receptors was obtained in the presence of divalent cations. Both the membrane-bound and the solubilized receptor activity were attenuated by guanylyl-imidodiphosphate (Gpp(NH)p) indicating that the brain CCKB receptors are coupled to G proteins.
Gut
S H
SH
MRC Molecular Neurobiology Unit, MRC Center, Cambridge, U.K.
Demoliou-Mason
C D
CD
Hunter
J C
JC
Hughes
J
J
Barnard
E A
EA
D000818:Animals; D001667:Binding, Competitive / Q000187:drug effects; D002412:Cations / Q000494:pharmacology; D002540:Cerebral Cortex / Q000187:drug effects / Q000378:metabolism*; D002766:Cholecystokinin / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D003902:Detergents; D004072:Digitonin; D006150:Guanine Nucleotides / Q000494:pharmacology; D066298:In Vitro Techniques; D007457:Iodine Radioisotopes; D007700:Kinetics; D008566:Membranes / Q000187:drug effects / Q000378:metabolism; D011949:Receptors, Cholecystokinin / Q000302:isolation & purification* / Q000378:metabolism; D012710:Serum Albumin, Bovine / Q000378:metabolism; D013552:Swine; D013816:Thermodynamics; D013997:Time Factors
D002412:Cations; D003902:Detergents; D006150:Guanine Nucleotides; D007457:Iodine Radioisotopes; D011949:Receptors, Cholecystokinin; D012710:Serum Albumin, Bovine; D002766:Cholecystokinin; D004072:Digitonin