Establishment of a new short, protease-resistant, affinity labeling reagent for the cholecystokinin receptor

Randall K. Pearson, Stephen P. Powers, Elizabeth M. Hadac, Herbert Gaisano, Laurence J Miller

Research output: Contribution to journalArticle

25 Citations (Scopus)

Abstract

Proteolytic degradation of radioligands is an important source of artifact in affinity labeling of receptor proteins. To complement our previous characterization of the pancreatic acinar cell cholecystokinin (CCK) receptor, we synthesized D-Tyr-Gly[(Nle28,31)CCK-26-33]. The amino terminal D-enantiomer of tyrosine provided a site for oxidative iodination, a free amino group for cross-linking and rendered the peptide resistant to aminopeptidases. The deca-peptide was oxidatively iodinated and purified by reverse-phase HPLC to 2,000 Ci/mmol, to yield a probe which was equal in potency and efficacy to CCK-8, and which bound to rat pancreatic membranes in a rapid, reversible, temperature-dependent, specific, saturable and high affinity manner. This probe was resistant to aminopeptidase degradation, and maintained its ability to bind to receptor after incubation with pancreatic membranes or dispersed cells. Affinity labeling of pancreatic membranes with this analogue identified an Mr=85,000-95,000 molecule. This analogue offers several advantages over existing probes and should be useful for future studies of this and other CCK receptors.

Original languageEnglish (US)
Pages (from-to)346-353
Number of pages8
JournalBiochemical and Biophysical Research Communications
Volume147
Issue number1
DOIs
StatePublished - Aug 31 1987

Fingerprint

Affinity Labels
Cholecystokinin Receptors
Aminopeptidases
Peptide Hydrolases
Membranes
Labeling
Degradation
Peptides
Enantiomers
Tyrosine
Rats
Acinar Cells
Halogenation
Artifacts
Molecules
High Pressure Liquid Chromatography
Temperature
Proteins

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

Cite this

Establishment of a new short, protease-resistant, affinity labeling reagent for the cholecystokinin receptor. / Pearson, Randall K.; Powers, Stephen P.; Hadac, Elizabeth M.; Gaisano, Herbert; Miller, Laurence J.

In: Biochemical and Biophysical Research Communications, Vol. 147, No. 1, 31.08.1987, p. 346-353.

Research output: Contribution to journalArticle

Pearson, Randall K. ; Powers, Stephen P. ; Hadac, Elizabeth M. ; Gaisano, Herbert ; Miller, Laurence J. / Establishment of a new short, protease-resistant, affinity labeling reagent for the cholecystokinin receptor. In: Biochemical and Biophysical Research Communications. 1987 ; Vol. 147, No. 1. pp. 346-353.
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