Bradykinin recognizes different molecular forms of the B2 kinin receptor in the presence and absence of guanine nucleotides
(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.
(Less)
- author
- Mathis, S. A. and Leeb-Lundberg, L. M.F. LU
- publishing date
- 1991-05-15
- type
- Contribution to journal
- publication status
- published
- subject
- in
- Biochemical Journal
- volume
- 276
- issue
- 1
- pages
- 141 - 147
- publisher
- Portland Press
- external identifiers
-
- scopus:0025856290
- pmid:1645526
- ISSN
- 0264-6021
- DOI
- 10.1042/bj2760141
- language
- English
- LU publication?
- no
- id
- 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}}, }