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Bradykinin recognizes different molecular forms of the B2 kinin receptor in the presence and absence of guanine nucleotides

Mathis, S. A. and Leeb-Lundberg, L. M.F. LU (1991) In Biochemical Journal 276(1). p.141-147
Abstract

We have previously reported that [3H]bradykinin ([3H]BK) identifies high- and low-affinity B2 kinin receptor sites in bovine myometrial membranes which are sensitive and insensitive respectively to guanine nucleotides. Here we show that these receptor-binding sites are solubilized by the detergent CHAPS. Equilibrium binding in soluble preparations revealed that [3H]BK identified a maximal number of binding sites (B(max)) of 119 ± 160 fmol/mg of protein, with an equilibrium dissociation constant (K(D)) of 314 ± 70 pM with a typical B2 kinin receptor specificity. Dissociation of equilibrium binding was biphasic. In the presence of the GTP analogue guanosine 5'[βγ-imido]triphosphate (Gpp[NH]p),... (More)

We have previously reported that [3H]bradykinin ([3H]BK) identifies high- and low-affinity B2 kinin receptor sites in bovine myometrial membranes which are sensitive and insensitive respectively to guanine nucleotides. Here we show that these receptor-binding sites are solubilized by the detergent CHAPS. Equilibrium binding in soluble preparations revealed that [3H]BK identified a maximal number of binding sites (B(max)) of 119 ± 160 fmol/mg of protein, with an equilibrium dissociation constant (K(D)) of 314 ± 70 pM with a typical B2 kinin receptor specificity. Dissociation of equilibrium binding was biphasic. In the presence of the GTP analogue guanosine 5'[βγ-imido]triphosphate (Gpp[NH]p), [3H]BK bound to the soluble receptors with a K(D) of 929 ± 129 pM and a B(max.) of 706 ± 38 fmol/mg of protein. The Gpp(NH)p-promoted decrease in the apparent affinity and B(max.), which was half-maximal at 0.5 μM, was due at least in part to an increase in the dissociation rate of the slowly dissociating component of the equilibrium binding. Recoveries of guanine-nucleotide-sensitivity and of rapidly and slowly dissociating binding components were essentially identical, whether or not the receptor had been occupied by an agonist before solubilization. Sucrose-density-gradient sedimentation profiles revealed that [3H]BK recognized two different molecular forms of the receptor in the absence or presence of guanine revealed that [3H]BK recognized two different molecular forms of the receptor in the absence or presence of guanine nucleotides. These results provide for the first time direct evidence that guanine nucleotides promote a change in the structure of the B2 kinin-receptor complex. We propose that this structural change is due to dissociation of a guanine-nucleotide-regulatory (G)-protein.

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Contribution to journal
publication status
published
subject
in
Biochemical Journal
volume
276
issue
1
pages
141 - 147
publisher
Portland Press
external identifiers
  • pmid:1645526
  • scopus:0025856290
ISSN
0264-6021
DOI
10.1042/bj2760141
language
English
LU publication?
no
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b71bec71-73a2-4cf1-a37a-bdfe24d9fb59
date added to LUP
2019-06-12 11:48:35
date last changed
2024-01-01 09:53:59
@article{b71bec71-73a2-4cf1-a37a-bdfe24d9fb59,
  abstract     = {{<p>We have previously reported that [<sup>3</sup>H]bradykinin ([<sup>3</sup>H]BK) identifies high- and low-affinity B2 kinin receptor sites in bovine myometrial membranes which are sensitive and insensitive respectively to guanine nucleotides. Here we show that these receptor-binding sites are solubilized by the detergent CHAPS. Equilibrium binding in soluble preparations revealed that [<sup>3</sup>H]BK identified a maximal number of binding sites (B(max)) of 119 ± 160 fmol/mg of protein, with an equilibrium dissociation constant (K(D)) of 314 ± 70 pM with a typical B2 kinin receptor specificity. Dissociation of equilibrium binding was biphasic. In the presence of the GTP analogue guanosine 5'[βγ-imido]triphosphate (Gpp[NH]p), [<sup>3</sup>H]BK bound to the soluble receptors with a K(D) of 929 ± 129 pM and a B(max.) of 706 ± 38 fmol/mg of protein. The Gpp(NH)p-promoted decrease in the apparent affinity and B(max.), which was half-maximal at 0.5 μM, was due at least in part to an increase in the dissociation rate of the slowly dissociating component of the equilibrium binding. Recoveries of guanine-nucleotide-sensitivity and of rapidly and slowly dissociating binding components were essentially identical, whether or not the receptor had been occupied by an agonist before solubilization. Sucrose-density-gradient sedimentation profiles revealed that [<sup>3</sup>H]BK recognized two different molecular forms of the receptor in the absence or presence of guanine revealed that [<sup>3</sup>H]BK recognized two different molecular forms of the receptor in the absence or presence of guanine nucleotides. These results provide for the first time direct evidence that guanine nucleotides promote a change in the structure of the B2 kinin-receptor complex. We propose that this structural change is due to dissociation of a guanine-nucleotide-regulatory (G)-protein.</p>}},
  author       = {{Mathis, S. A. and Leeb-Lundberg, L. M.F.}},
  issn         = {{0264-6021}},
  language     = {{eng}},
  month        = {{05}},
  number       = {{1}},
  pages        = {{141--147}},
  publisher    = {{Portland Press}},
  series       = {{Biochemical Journal}},
  title        = {{Bradykinin recognizes different molecular forms of the B2 kinin receptor in the presence and absence of guanine nucleotides}},
  url          = {{http://dx.doi.org/10.1042/bj2760141}},
  doi          = {{10.1042/bj2760141}},
  volume       = {{276}},
  year         = {{1991}},
}