Cholecystokinin (CCK) is a peptide hormone that has a variety of physiologically important functions in the gastrointestinal tract, in which distinct high affinity receptors have been identified. We describe here the purification of the digitonin-solubilized rat pancreatic receptor as an initial step in the determination of its primary structure. Solubilization of total pancreatic membranes using 1% digitonin resulted in a single class of binding sites with a specific content of 4 pmol/mg as measured in soluble binding assay using the nonpeptidyl CCK antagonist [3H]3S[-]-N-[2,3-dihydro-1-methyl-2-oxo-5-phenyl-1H-1,4-benzodiazepin e-3-yl]-1H-indole-2-carboxamide ([3H]364,718). The solubilized receptor was purified using the following chromatographic steps: 1) cation exchange; 2) Ulex europaeus agglutinin-I-agarose; and 3) Sephacryl S-300. The final preparation of the purified receptor had a specific content of 8,055 pmol/mg, which represented a 9,051-fold purification from intact membranes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the purified receptor preparation under reducing conditions resulted in a predominant polypeptide with an M(r) = 85,000-95,000 and minor polypeptides of M(r) = 57,000 and 26,000 as determined by radiolabeling and silver staining. Solubilized pancreatic membranes were affinity labeled with the peptidyl CCK agonist 125I-D-Tyr-Gly-[(Nle28,31,6-NO2-Phe33)CCK-26-33] and chromatographed under conditions similar to those described for untreated membranes. Elution of radioactive peaks from each chromatographic column was coincident with [3H]364,718 binding activity and resulted in a labeled polypeptide having the same electrophoretic mobility as receptor derived from freshly labeled membranes and purified from untreated membranes. High performance liquid-gel exclusion chromatography of the crude digitonin-solubilized membrane preparation revealed an estimated molecular size for the [3H]364,718-binding activity of 370,000, which was consistent with the size determined by nondenaturing gel electrophoresis of the purified receptor complexed with the labeled nonpeptidyl antagonist. Binding of [3H]364,718 to the purified receptor preparation was comparable to that observed with the crude solubilized pancreatic membrane preparation; and both the homologous ligand 364,718 (K(i) = 0.5 nm) and CCK-8 (K(i) = 1.4 μM) competed for binding to both preparations in a similar manner.
|Original language||English (US)|
|Number of pages||7|
|Journal||Journal of Biological Chemistry|
|State||Published - Jan 1 1989|
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology