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 language | English (US) |
|---|---|
| Journal | American Journal of Physiology - Gastrointestinal and Liver Physiology |
| Volume | 258 |
| Issue number | 5 21-5 |
| State | Published - 1990 |
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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 journal › Article
}
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
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 -