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(Received for publication, May 18, 1995; and in revised form, June 14, 1995)
Chemical cross-linking was used to analyze the binding sites for
the agonist bradykinin (BK) and the antagonists NPC17731 and HOE140 on
the bovine B2 bradykinin receptor. [
H]BK and
[H]NPC17731 bound with high affinity to the same
B2 receptor in bovine myometrial membranes as determined by the total
number of specific binding sites and pharmacological specificity of the
binding of these two radioligands. Cross-linking experiments were done
using a series of bifunctional reagents reactive either primarily to
amines (homobifunctional) or reactive to amines in one end and to
sulfhydryls in the opposite end (heterobifunctional). All the
heterobifunctional reagents plus the homobifunctional arylhalide
1,5-difluoro-2,4-dinitrobenzene were effective in cross-linking the
[H]BK N terminus specifically to a sulfhydryl in
the receptor, and this cross-linking occurred at 5-100 µM reagent. In contrast, the homobifunctional N-hydroxysuccinimide ester reagents, at 
1 mM,
were only able to cross-link [H]BK to membrane
proteins nonspecifically. The sulfhydryl reagents N-ethylmaleimide, iodoacetamide, and phenylarsine oxide
blocked cross-linking, whereas these reagents did not inhibit
reversible specific [H]BK binding. Immunoblotting
with anti-BK antiserum revealed that low concentrations of BK
(5-50 nM) were cross-linked to a receptor-specific
species of 65 kDa. All cross-linking of
[H]NPC17731 was nonspecific with both
homobifunctional and heterobifunctional reagents. The 65-kDa
receptor-specific species was observed on anti-HOE140 immunoblots, but
only when proteins were cross-linked with very high concentrations of
HOE140 (
500 nM). Our results provide direct biochemical
evidence that the binding site for the agonist BK in the bovine B2
receptor is adjacent to a cysteine and is differentiated from the
binding site(s) for the antagonists NPC17731 and HOE140.
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