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.[1]References
- Preparation of enriched plasma membranes from bovine gallbladder muscularis for characterization of cholecystokinin receptors. Shaw, M.J., Hadac, E.M., Miller, L.J. J. Biol. Chem. (1987) [Pubmed]
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