Herzig M C, Leeb-Lundberg L M
Department of Biochemistry, University of Texas Health Science Center, San Antonio 78284-7760, USA.
J Biol Chem. 1995 Sep 1;270(35):20591-8. doi: 10.1074/jbc.270.35.20591.
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. [3H]BK and [3H]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 [3H]BK N terminus specifically to a sulfhydryl in the receptor, and this cross-linking occurred at 5-100 microM reagent. In contrast, the homobifunctional N-hydroxysuccinimide ester reagents, at < or = 1 mM, were only able to cross-link [3H]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 [3H]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 [3H]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 (> or = 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.
采用化学交联法分析激动剂缓激肽（BK）以及拮抗剂NPC17731和HOE140在牛B2缓激肽受体上的结合位点。通过特异性结合位点总数以及这两种放射性配体结合的药理学特异性测定，[3H]BK和[3H]NPC17731以高亲和力结合于牛子宫肌层膜中的同一B2受体。交联实验使用了一系列双功能试剂，这些试剂要么主要与胺反应（同型双功能），要么一端与胺反应而另一端与巯基反应（异型双功能）。所有异型双功能试剂加上同型双功能芳基卤化物1,5-二氟-2,4-二硝基苯均能有效地将[3H]BK的N末端特异性交联至受体中的一个巯基，且这种交联在5 - 100μM试剂浓度下发生。相比之下，浓度≤1 mM的同型双功能N-羟基琥珀酰亚胺酯试剂仅能非特异性地将[3H]BK交联至膜蛋白。巯基试剂N-乙基马来酰亚胺、碘乙酰胺和苯胂氧化物可阻断交联，而这些试剂并不抑制[3H]BK的可逆特异性结合。用抗BK抗血清进行免疫印迹显示，低浓度的BK（5 - 50 nM）与一种65 kDa的受体特异性蛋白交联。[3H]NPC17731的所有交联在用同型双功能和异型双功能试剂时均为非特异性。在抗HOE140免疫印迹上观察到了65 kDa的受体特异性蛋白，但仅当蛋白与非常高浓度的HOE140（≥500 nM）交联时才会出现。我们的结果提供了直接的生化证据，表明牛B2受体中激动剂BK的结合位点与一个半胱氨酸相邻，且与拮抗剂NPC17731和HOE140的结合位点不同。
