Purification and characterization of the rat pancreatic cholecystokinin receptor

L. T. Duong, E. M. Hadac, Laurence J Miller, G. P. Vlasuk

Research output: Contribution to journalArticle

29 Citations (Scopus)

Abstract

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 languageEnglish (US)
Pages (from-to)17990-17996
Number of pages7
JournalJournal of Biological Chemistry
Volume264
Issue number30
StatePublished - 1989
Externally publishedYes

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Cholecystokinin Receptors
Purification
Rats
Membranes
Digitonin
Cholecystokinin
Electrophoresis
Peptides
Gel Chromatography
Gels
Ulex
Electrophoretic mobility
Silver Staining
Peptide Hormones
Agglutinins
Chromatography
Silver
Sodium Dodecyl Sulfate
Sepharose
Gastrointestinal Tract

ASJC Scopus subject areas

  • Biochemistry

Cite this

Purification and characterization of the rat pancreatic cholecystokinin receptor. / Duong, L. T.; Hadac, E. M.; Miller, Laurence J; Vlasuk, G. P.

In: Journal of Biological Chemistry, Vol. 264, No. 30, 1989, p. 17990-17996.

Research output: Contribution to journalArticle

Duong, L. T. ; Hadac, E. M. ; Miller, Laurence J ; Vlasuk, G. P. / Purification and characterization of the rat pancreatic cholecystokinin receptor. In: Journal of Biological Chemistry. 1989 ; Vol. 264, No. 30. pp. 17990-17996.
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