Preparation of enriched plasma membranes from bovine gallbladder muscularis for characterization of cholecystokinin receptors.
A method for the preparation of enriched plasma membranes from bovine gallbladder muscularis was developed, validated, and applied to the characterization of receptors for the gastrointestinal hormone cholecystokinin (CCK) on this target. Binding of radioiodinated CCK ligands to this preparation was rapid, reversible, temperature-dependent, saturable, and specific. Only structurally related peptides inhibited CCK binding, and good correlation existed between relative potencies for binding inhibition and for stimulating gallbladder contraction. Computer analysis of CCK-binding data using a nonlinear model-fitting program best fit a model with a single class of sites, with Kd 756 pm and binding capacity 4.5 +/- 1.3 pmol/mg of protein. This degree of enrichment for plasma membranes was adequate for the initial biochemical characterization of this CCK receptor. Affinity labeling using 125I-Bolton Hunter-CCK-33 and m-maleimidobenzoyl-N-hydroxysuccinimide ester identified proteins with Mr = 70,000-85,000, Mr = 120,000-125,000, and Mr = 200,000. Labeling was inhibited in a concentration-dependent manner, with an IC50 of 1 nM CCK-8, and the electrophoretic mobility of these bands was not different under reducing and nonreducing conditions. The major labeled band of Mr = 70,000-85,000 has a lower apparent Mr than that of the analogous band in pancreas labeled with similar methods, supporting the molecular heterogeneity of CCK receptors on these two target tissues.
Shaw
M J
MJ
Gastroenterology Research Unit, Mayo Clinic and Foundation, Rochester, Minnesota 55905.
Hadac
E M
EM
Miller
L J
LJ
D000818:Animals; D002417:Cattle; D002458:Cell Fractionation; D002462:Cell Membrane / Q000378:metabolism / Q000648:ultrastructure; D002499:Centrifugation, Density Gradient / Q000379:methods; D005704:Gallbladder / Q000378:metabolism*; D007700:Kinetics; D008970:Molecular Weight; D009130:Muscle, Smooth / Q000378:metabolism; D011949:Receptors, Cholecystokinin / Q000302:isolation & purification / Q000378:metabolism*; D013816:Thermodynamics
D011949:Receptors, Cholecystokinin