Identification and localization of cholecystokinin-binding sites on rat pancreatic plasma membranes and acinar cells: a biochemical and autoradiographic study.

Abstract

Using the combined approaches of affinity labeling, light microscopic and EM autoradiography, the identification and localization of cholecystokinin (CCK)-binding sites were investigated on rat pancreatic acinar cells. To define the molecular properties of the CCK-binding site, rat pancreatic plasma membranes were incubated with 125I-CCK-33 for 15 min at 23.degree. C followed by washing and cross-linking with disuccinimidyl suberate. Specific labeling of a major MW 85,000 component was revealed as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions and autoradiography of the dried gels. Components of MW > 200,000, MW 130,000-140,000, and MW 55,000 were labeled under maximal cross-linking conditions. The labeling of all components was specifically inhibited by CCK-8 in a dose-dependent manner (Kd .apprx. 9 nM). The MW 85,000 component had identical electrophoretic mobilities under reducing and nonreducing conditions, indicating that it likely does not contain intramolecular disulfide bonds. The larger labeled species may be cross-linked oligomers of this binding protein or complexes between it and neighboring polypeptides. For studies on the distribution of CCK-binding sites, pancreatic acini were incubated with 125I-CCK-33 (0.1 nM) in the absence or presence of CCK-8 (1 .mu.M) for 2 or 15 min at 37.degree. C, washed, and fixed in 2% glutaraldehyde. Quantitative autoradiographic analysis indicated that .apprx. 60% of the total grains were located within .+-. 1 HD (1 HD = 100 nm) of the lateral and basal plasmalemma with little or no labeling of the apical plasmalemma. Each acinar cell apparently possesses at least 5000-10,000 CCK-binding sites on its basolateral plasmalemma. The remaining grains showed no preferential concentration over the cytoplasm or nucleus. CCK evidently interacts with a MW 85,000 protein located on the basolateral plasmalemma of the pancreatic acinar cell.