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Biochemical characterization of the pancreatic cholecystokinin receptor using monofunctional photoactivatable probes

Journal Article · · Pancreas; (United States)
; ; ;

Receptor characterization by affinity labeling can be enhanced by taking multiple complementary approaches. To extend our observations on the subunit structure of the rat pancreatic cholecystokinin (CCK) receptor (made using bifunctional cross-linking reagents), we synthesized two monofunctional photoactivatable receptor probes. CCK-8 was acylated with the iodinated aryl azide derivatives, methyl-3-azido-4-hydroxy-5-(/sup 125/I)iodobenzimidate and N-(4-(4'-azido-3'-(/sup 125/I)iodophenylazo)benzoyl)-3-aminopropionyl-N- oxy- succinimide. The products were purified by reverse-phase HPLC to a specific radioactivity of 2000 Ci/mmol. Both analogs demonstrated saturable and specific binding to rat pancreatic plasma membranes. Photoaffinity labeling of pancreatic membranes with these monofunctional probes identified an Mr 85,000-95,000 protein that was not part of a larger disulfide-linked complex. High affinity for CCK was demonstrated by the concentration-dependent inhibition of labeling observed with competing CCK-8 (IC50 = 1 nM). On sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) this protein co-migrates with the major component we identified using a series of cross-linkable, iodinated decapeptide analogs of CCK, and is different from the major protein labeled using /sup 125/I-Bolton Hunter-CCK-33. Thus, these results support the presence of an Mr 85,000-95,000 subunit in the pancreatic CCK receptor, while the small size of these photoaffinity probes and their monovalency suggest that this subunit may contain or be spatially apposed to the active binding site. These probes should be very useful in the further characterization of this and other receptors for this hormone.

Research Organization:
Mayo Clinic, Rochester, MN
OSTI ID:
6501249
Journal Information:
Pancreas; (United States), Journal Name: Pancreas; (United States) Vol. 1; ISSN PANCE
Country of Publication:
United States
Language:
English

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Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
AFFINITY
ANIMALS
AZIDES
BETA DECAY RADIOISOTOPES
BIOCHEMICAL REACTION KINETICS
BODY
CELL CONSTITUENTS
CELL MEMBRANES
CHEMICAL COMPOSITION
CHEMICAL REACTIONS
CHEMISTRY
CHROMATOGRAPHY
CROSS-LINKING
DAYS LIVING RADIOISOTOPES
DIGESTIVE SYSTEM
ELECTRON CAPTURE RADIOISOTOPES
ELECTROPHORESIS
ENDOCRINE GLANDS
GLANDS
INTERMEDIATE MASS NUCLEI
IODINE 125
IODINE ISOTOPES
ISOTOPE APPLICATIONS
ISOTOPES
KINETICS
KININS
LIQUID COLUMN CHROMATOGRAPHY
MAMMALS
MEMBRANE PROTEINS
MEMBRANES
MOLECULAR STRUCTURE
NITROGEN COMPOUNDS
NUCLEI
ODD-EVEN NUCLEI
ORGANIC COMPOUNDS
ORGANS
PANCREAS
PEPTIDES
PHOTOCHEMISTRY
POLYMERIZATION
POLYPEPTIDES
PROTEINS
RADIOISOTOPES
RATS
REACTION KINETICS
RECEPTORS
RODENTS
SEPARATION PROCESSES
TRACER TECHNIQUES
VERTEBRATES