| Abstract: | In order to characterize the CCK receptor in guinea-pig pancreas, iodinated CCK-39 was bound to pancreatic membranes and the reversible complex was solubilized using various non-denaturing detergents. In term of recovery of ligand stabilized receptors, the relative potencies were Zwittergent 3-14 greater than CHAPS = CHAPSO greater than digitonin greater than MEGA 10 greater than octyl beta-D-glucopyranoside. The stability of receptor complexes was increased by glycerol. Chromatographic analysis revealed that digitonin was the most efficient detergent for disaggregation of CCK receptor complex since it yielded a 76 kDa component in addition to the large components obtained after solubilization with CHAPS and Zwittergent. Furthermore, CCK receptors were covalently labelled using dissuccinimidyl suberate or UV irradiation of labelled membranes by photoactivable radioiodinated CCK-39 and subsequently solubilized by CHAPS + SDS or by SDS alone. A predominant molecule was characterized by chromatography (76 kDa) and SDS-PAGE (89 kDa). In addition to this component, other components having molecular masses of 130-150 kDa, 57 kDa and 40 kDa were detected by SDS-PAGE. They correspond to minor bands. These bands, except the 40 kDa band, were protected from covalent labelling by the presence of CCK-39 (10(-6) M) during initial incubation. Reduction under beta-mercaptoethanol mainly resulted in the decrease of high molecular weight aggregates (Mr greater than 200 kDa). We concluded that for a given detergent a specific molecular weight pattern of solubilized CCK receptor complex is achieved. The minimal component had a molecular mass of 71-84 kDa according to the method of biochemical analysis used. |
