High-affinity binding sites for bombesin on mouse colonic mucosal membranes

Satya Narayan, Edwin Draviam, Srinivasan Rajaraman, Pomila Singh

Research output: Contribution to journalArticle

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Abstract

Bombesin (BBS) has specific biological effects on colonic mucosal cells, but the presence of BBS receptors on colonic mucosa have not been described to-date. In the present study we examined the mouse colonic mucosal membranes for the presence of specific binding sites for BBS/gastrin releasing peptides (GRP), and characterized the binding kinetics and molecular weight of the specific binding proteins. The radiolabeled ligand (125I-Tyr4-BBS), in the absence or presence of a 1000-fold excess of BBS, was used to establish the optimal binding assay conditions of time, pH and temperature for measuring the maximum number of specific binding sites for BBS related peptides. Under the optimal binding assay conditions, BBS displaced the binding of 125I-Tyr4-BBS in a dose-related manner. A single class of high-affinity binding sites (Kd = 0.23 ±0.02 nM) for BBS were measured, with a binding capacity of 27.3 + 4.6 fmoles/mg membrane protein. The binding sites were specific for binding BBS/GRP related peptides, since all structurally related peptides inhibited the binding of 125I-Tyro-BBS in a dose-dependent manner, while structurally unrelated peptides did not compete for the 125I-Tyro-BBS binding sites. The relative binding affinity (RBA) of BBS/GRP related peptides was determined to be in the order of GRP (14-27) = GRP (18-27) > GRP (1-27) > neuromedin B > BBS. The BBS-receptor antagonists, [Leu13-ψ-(CH2NH) Lcu14]-BBS (LL-BBS) and D-Phe6, BN(6-13) propylamide (D-Phe6,BN(6-13)-PA), inhibited the specific binding of 125I-Tyr4-BBS to colonic mucosal membranes in a dose-dependent manner. The inhibitory potency of D-Phe6,BN[6-13]PA was significantly greater than that of LL-1313S. Molecular mass of the specific binding proteins for BBS/GRP was determined to be 70-80 KDa, by chemical cross-linking methods. The 70-80 KDa binding proteins were specific for binding GRP-related peptides and were displaced in dose-dependent manner by increasing doses of BBS. These results thus suggest that the colonic mucosa may be yet another target for GRP related peptides.

Original languageEnglish (US)
Pages (from-to)31-39
Number of pages9
JournalMolecular and Cellular Biochemistry
Volume106
Issue number1
DOIs
StatePublished - Jul 1991

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Bombesin
Binding Sites
Membranes
Gastrin-Releasing Peptide
Peptides
Bombesin Receptors
Carrier Proteins
Assays
Mucous Membrane

Keywords

  • bombesin receptors
  • gastrin releasing peptides
  • moise colon mucosa

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics
  • Clinical Biochemistry
  • Cell Biology

Cite this

High-affinity binding sites for bombesin on mouse colonic mucosal membranes. / Narayan, Satya; Draviam, Edwin; Rajaraman, Srinivasan; Singh, Pomila.

In: Molecular and Cellular Biochemistry, Vol. 106, No. 1, 07.1991, p. 31-39.

Research output: Contribution to journalArticle

Narayan, Satya ; Draviam, Edwin ; Rajaraman, Srinivasan ; Singh, Pomila. / High-affinity binding sites for bombesin on mouse colonic mucosal membranes. In: Molecular and Cellular Biochemistry. 1991 ; Vol. 106, No. 1. pp. 31-39.
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abstract = "Bombesin (BBS) has specific biological effects on colonic mucosal cells, but the presence of BBS receptors on colonic mucosa have not been described to-date. In the present study we examined the mouse colonic mucosal membranes for the presence of specific binding sites for BBS/gastrin releasing peptides (GRP), and characterized the binding kinetics and molecular weight of the specific binding proteins. The radiolabeled ligand (125I-Tyr4-BBS), in the absence or presence of a 1000-fold excess of BBS, was used to establish the optimal binding assay conditions of time, pH and temperature for measuring the maximum number of specific binding sites for BBS related peptides. Under the optimal binding assay conditions, BBS displaced the binding of 125I-Tyr4-BBS in a dose-related manner. A single class of high-affinity binding sites (Kd = 0.23 ±0.02 nM) for BBS were measured, with a binding capacity of 27.3 + 4.6 fmoles/mg membrane protein. The binding sites were specific for binding BBS/GRP related peptides, since all structurally related peptides inhibited the binding of 125I-Tyro-BBS in a dose-dependent manner, while structurally unrelated peptides did not compete for the 125I-Tyro-BBS binding sites. The relative binding affinity (RBA) of BBS/GRP related peptides was determined to be in the order of GRP (14-27) = GRP (18-27) > GRP (1-27) > neuromedin B > BBS. The BBS-receptor antagonists, [Leu13-ψ-(CH2NH) Lcu14]-BBS (LL-BBS) and D-Phe6, BN(6-13) propylamide (D-Phe6,BN(6-13)-PA), inhibited the specific binding of 125I-Tyr4-BBS to colonic mucosal membranes in a dose-dependent manner. The inhibitory potency of D-Phe6,BN[6-13]PA was significantly greater than that of LL-1313S. Molecular mass of the specific binding proteins for BBS/GRP was determined to be 70-80 KDa, by chemical cross-linking methods. The 70-80 KDa binding proteins were specific for binding GRP-related peptides and were displaced in dose-dependent manner by increasing doses of BBS. These results thus suggest that the colonic mucosa may be yet another target for GRP related peptides.",
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N2 - Bombesin (BBS) has specific biological effects on colonic mucosal cells, but the presence of BBS receptors on colonic mucosa have not been described to-date. In the present study we examined the mouse colonic mucosal membranes for the presence of specific binding sites for BBS/gastrin releasing peptides (GRP), and characterized the binding kinetics and molecular weight of the specific binding proteins. The radiolabeled ligand (125I-Tyr4-BBS), in the absence or presence of a 1000-fold excess of BBS, was used to establish the optimal binding assay conditions of time, pH and temperature for measuring the maximum number of specific binding sites for BBS related peptides. Under the optimal binding assay conditions, BBS displaced the binding of 125I-Tyr4-BBS in a dose-related manner. A single class of high-affinity binding sites (Kd = 0.23 ±0.02 nM) for BBS were measured, with a binding capacity of 27.3 + 4.6 fmoles/mg membrane protein. The binding sites were specific for binding BBS/GRP related peptides, since all structurally related peptides inhibited the binding of 125I-Tyro-BBS in a dose-dependent manner, while structurally unrelated peptides did not compete for the 125I-Tyro-BBS binding sites. The relative binding affinity (RBA) of BBS/GRP related peptides was determined to be in the order of GRP (14-27) = GRP (18-27) > GRP (1-27) > neuromedin B > BBS. The BBS-receptor antagonists, [Leu13-ψ-(CH2NH) Lcu14]-BBS (LL-BBS) and D-Phe6, BN(6-13) propylamide (D-Phe6,BN(6-13)-PA), inhibited the specific binding of 125I-Tyr4-BBS to colonic mucosal membranes in a dose-dependent manner. The inhibitory potency of D-Phe6,BN[6-13]PA was significantly greater than that of LL-1313S. Molecular mass of the specific binding proteins for BBS/GRP was determined to be 70-80 KDa, by chemical cross-linking methods. The 70-80 KDa binding proteins were specific for binding GRP-related peptides and were displaced in dose-dependent manner by increasing doses of BBS. These results thus suggest that the colonic mucosa may be yet another target for GRP related peptides.

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