Molecular characterization of bombesin receptors on rat pancreatic acinar AR42J cells

P. Singh, E. Draviam, Y. S. Guo, A. Kurosky

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Abstract

A biologically active, chemically defined, radioactive ligand was used for characterizing bombesin (BBS) receptors on rat pancreatic acinar cells (AR42J). [Tyr4]BBS, iodinated with enzymobeads and fractionated by high-performance liquid chromatography, was monitored for biological activity as evidenced by gastrin release from perfused isolated rat stomach. The monoiodinated peptide peak was >95% biologically active, with a specific activity of >2,000 disintegrations·min-1·fmol-1. The maximum number of BBS receptors per cell were measured at 30°C after 20-25 min of incubation; binding was submaximum at temperatures lower or higher than 30°C. A single class of high-affinity binding sites (K(d) = 1.77 ± 0.21 nM) was identified for BBS on AR42J cells and nonspecific binding was <20-30% at all points. A total of 1.47 ± 0.14 x 105 specific BBS binding sites per cell were measured that were specific for BBS and gastrin-releasing peptide (GRP) analogues. Iodinated GRP-(1-27) was cross-linked to BBS receptors on AR42J cells using several bifunctional cross-linking reagents followed by polyacrylamide gel electrophoresis of the solubilized receptor complex under reducing and nonreducing conditions. A densitometric analysis of the autoradiographs demonstrated the presence of an ~80- to 85-kDa molecular form of the receptor as a major component under both reducing and nonreducing conditions. These results indicated that the receptor molecule is a single subunit without multiple chains covalently attached by disulfide bonding.

Original languageEnglish (US)
Pages (from-to)G803-G809
JournalAmerican Journal of Physiology - Gastrointestinal and Liver Physiology
Volume258
Issue number5 21-5
StatePublished - Jan 1 1990

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Keywords

  • bombesin binding to AR42J cells
  • gastrin-releasing peptide

ASJC Scopus subject areas

  • Physiology
  • Hepatology
  • Gastroenterology
  • Physiology (medical)

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