Evidence of essential disulfide bonds in angiotensin II binding sites of rabbit hepatic membranes. Inactivation by dithiothreitol.
Evidence of essential disulfide bonds in angiotensin II binding sites of rabbit hepatic membranes. Inactivation by dithiothreitol.
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兔肝膜血管紧张素 II 结合位点中必需二硫键的证据。
DOI：
10.1016/0005-2736(85)90350-5
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发表时间：
1985
期刊：
影响因子：
--
通讯作者：
Sen,I
中科院分区：
文献类型：
--
作者：
Sen,I
放射性标记的血管紧张素II结合到纯化的兔肝膜上的一类高亲和力结合位点。结合是特异性的、可逆的和可饱和的。使用血管紧张素和血管紧张素II的各种类似物的置换研究公开了与生理学相关的血管紧张素II受体位点中发现的类似的结构-活性特征。用还原剂二硫苏糖醇处理膜，导致血管紧张素II结合位点对天然配体的亲和力显著降低。该效应通过高15倍浓度的单巯基衍生物2-巯基乙醇来模拟。动力学研究还表明，二硫苏糖醇增加结合配体从膜上解离的速率，而不显着影响缔合速率。相反，用金属螯合剂乙二胺四乙酸（EDTA）和乙二醇双（β-氨基乙基醚）-N，N′-四乙酸（EGTA）处理膜，不影响放射性标记的血管紧张素II的结合。此外，二硫苏糖醇抑制血管紧张素II的结合，从同一组织的血管紧张素II结合蛋白的溶解的部分纯化的制剂，也增加了结合的血管紧张素II的解离。这表明巯基试剂对膜结合位点的影响是直接改变结合位点的结果，而不是膜结构的总体改变。
Radiolabelled angiotensin II binds to a single class of high-affinity binding sites on purified rabbit hepatic membranes. The binding is specific, reversible and saturable. Displacement studies using angiotensin and various analogs of angiotensin II disclosed a structure-activity profile similar to that found in physiologically relevant angiotensin II receptor sites. Treatment of membranes with the reducing agent, dithiothreitol, cause a significant decrease in the affinity of angiotensin II binding sites for the native ligand. This effect is mimicked by a 15-fold higher concentration of the monosulfhydryl derivative, 2-mercaptoethanol. Kinetic studies also indicated that dithiothreitol increases the rate of dissociation of bound ligand from the membrane without significantly affecting the association rate. In contrast, treatment of membranes with the metal chelators, ethylenediaminetetracetic acid (EDTA) and ethyleneglycol bis(β-aminoethyl ether)-N,N′-tetracetic acid (EGTA), does not affect the binding of radiolabeled angiotensin II. Furthermore, dithiothreitol inhibited the binding of angiotensin II to a solubilized partially purified preparation of angiotensin II-binding protein from the same tissue and also increased the dissociation of bound angiotensin II. This indicates that the effect of the sulfhydryl reagents on the membrane binding sites is the result of a direct alteration of the binding sites rather than a gross modification of the structure of the membrane.