Molecular characterization of bombesin receptors on rat pancreatic acinar AR42J cells

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

Research output: Contribution to journalArticle

27 Citations (Scopus)

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)
JournalAmerican Journal of Physiology - Gastrointestinal and Liver Physiology
Volume258
Issue number5 21-5
StatePublished - 1990

Fingerprint

Bombesin Receptors
Bombesin
Acinar Cells
Gastrin-Releasing Peptide
Cross-Linking Reagents
Binding Sites
Gastrins
Disulfides
Polyacrylamide Gel Electrophoresis
Stomach
High Pressure Liquid Chromatography
Ligands
Peptides
Temperature

Keywords

  • bombesin binding to AR42J cells
  • gastrin-releasing peptide

ASJC Scopus subject areas

  • Physiology
  • Gastroenterology

Cite this

Molecular characterization of bombesin receptors on rat pancreatic acinar AR42J cells. / Singh, Pomila; Draviam, E.; Guo, Y. S.; Kurosky, A.

In: American Journal of Physiology - Gastrointestinal and Liver Physiology, Vol. 258, No. 5 21-5, 1990.

Research output: Contribution to journalArticle

@article{403c043053974a368b4925160880f50a,
title = "Molecular characterization of bombesin receptors on rat pancreatic acinar AR42J cells",
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.",
keywords = "bombesin binding to AR42J cells, gastrin-releasing peptide",
author = "Pomila Singh and E. Draviam and Guo, {Y. S.} and A. Kurosky",
year = "1990",
language = "English (US)",
volume = "258",
journal = "American Journal of Physiology - Endocrinology and Metabolism",
issn = "0193-1849",
publisher = "American Physiological Society",
number = "5 21-5",

}

TY - JOUR

T1 - Molecular characterization of bombesin receptors on rat pancreatic acinar AR42J cells

AU - Singh, Pomila

AU - Draviam, E.

AU - Guo, Y. S.

AU - Kurosky, A.

PY - 1990

Y1 - 1990

N2 - 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.

AB - 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.

KW - bombesin binding to AR42J cells

KW - gastrin-releasing peptide

UR - http://www.scopus.com/inward/record.url?scp=0025354209&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0025354209&partnerID=8YFLogxK

M3 - Article

C2 - 2159242

AN - SCOPUS:0025354209

VL - 258

JO - American Journal of Physiology - Endocrinology and Metabolism

JF - American Journal of Physiology - Endocrinology and Metabolism

SN - 0193-1849

IS - 5 21-5

ER -