Effects of SH-modifying reagents on the rat hepatic Ah receptor: inhibition of ligand binding and transformation, and disruption of the ligand-receptor complex.
Effects of SH-modifying reagents on the rat hepatic Ah receptor: inhibition of ligand binding and transformation, and disruption of the ligand-receptor complex.
复制标题
SH 修饰试剂对大鼠肝 Ah 受体的影响：抑制配体结合和转化，破坏配体-受体复合物。
DOI：
10.1016/0304-4165(88)90037-2
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发表时间：
1988
期刊：
影响因子：
--
通讯作者：
Gasiewicz,TA
中科院分区：
文献类型：
--
作者：
Henry,EC;Kester,JE;Gasiewicz,TA
用各种巯基(SH)修饰试剂证明了巯基(SH)在维持大鼠肝细胞受体的物理化学性质中的重要性。对2，3，7，8-四氯二苯并-对二恶英的抑制作用与5，5‘-二硫双(2-硝基苯甲酸酯)、山梨醇、N-乙基马来酰亚胺和对氯汞苯磺酸盐大致相当，其抑制曲线在接近胞浆中非蛋白SH基团的浓度范围内陡峭(ED50值为50~200nmoL M或13~48nmoL/mg胞浆蛋白)。抑制BYP-羟基汞苯甲酸盐(PHMB)，虽然表现出较低的ED50，在这个范围内是更渐进的；碘乙酰胺的效力要小一个数量级。二硫苏糖醇逆转150μM(约60nmol/mg蛋白质)Mersalyl引起的结合抑制的能力随时间而减弱；TCDD和Mersalyl同时存在时，其下降速度比单独存在时更快，这与TCDD受体复合体形成过程中构象变化增加关键SH基团(S)的可达性一致。在TCDD结合之前，将未被占据的受体短暂暴露于Mersalyl，导致该复合体在O-KCl蔗糖梯度中的沉积变慢，并且其DEAE-和DNA-Spharose洗脱谱的改变暗示着转化过程的一些损害。当将试剂添加到转化的TCDD-受体复合体中时，只有在比抑制TCDD结合的浓度高一个数量级的浓度下才能观察到结合的丧失。除Mersalyl引起的结合丧失外，每种试剂的结合丧失都是两相的，即使在6-8h后仍不完全，二硫苏糖醇只能部分逆转Mersalyl或PHMB的作用，且只能在结合丧失的早期阶段(10-30min)加入。与转化的TCDD受体复合体相比，Mersalyl对未转化的TCDD受体复合体的破坏作用要强得多。经Mersalyl处理的TCDD受体复合体的物理变化从凝胶过滤、蔗糖梯度以及DNA和DEAE-Sepharose层析中明显可见。我们的结果与这些试剂对类固醇受体的影响形成鲜明对比，类固醇受体结合的类固醇激素配体被较低浓度的试剂迅速和可逆地取代。观察到的结果是一致的：(1)Mersalyl引起不可逆的改变，如未被占据的Ah受体以及受体-配体复合体的聚集和/或变性；(2)与未被转化的复合体和未被占据的受体相比，转化的TCDD-受体复合体对这些SH受体的破坏的敏感性降低(即，配体结合和转化引起受体的变化，这可能改变SH基团的可及性)。
The importance of sulfhydryl (SH) groups in maintenance of physicochemical properties of the rat hepaticAhreceptor was demonstrated using a variety of sulfhydryl (SH)-modifying reagents. Inhibition of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) specific binding was approximately equivalent by 5,5′-dithiobis(2-nitrobenzoate), mersalyl,N-ethylmaleimide, andp-chloromercuriphenylsulfonate, whose inhibition curves were steep in the concentration range close to that of nonprotein SH groups in cytosol (ED50values 50–200 μM or 13–48 nmol/mg cytosolic protein). Inhibition byp-hydroxymercuribenzoate (PHMB), although exhibiting a lower ED50, was more gradual over this range; iodoacetamide was an order of magnitude less potent. The ability of dithiothreitol to reverse binding inhibition induced by 150 μM (approximately 60 nmol/mg protein) mersalyl diminished with time; it decreased more rapidly in the simultaneous presence of TCDD and mersalyl than when mersalyl was present alone, consistent with increased accessibility of key SH group(s) due to conformational changes attending TCDD-receptor complex formation. Brief exposure of unoccupied receptor to mersalyl prior to TCDD binding caused slower sedimentation of the complex in O-KCl sucrose gradients and alterations in its elution profiles of DEAE- and DNA-Spharose suggestive of some impairment of the transformation process. When reagents were added to the transformed TCDD-receptor complex, loss of binding was observed only at concentrations which were an order of magnitude higher than those inhibiting TCDD binding. Loss of binding by each reagent was biphasic, and except for that caused by mersalyl, was not complete even after 6–8 h. Dithiothreitol was able to reverse the effects of mersalyl or PHMB only partially and only if added during the early phase (10–30 min) of binding loss. Mersalyl was much more potent in disrupting the untransformed than the transformed TCDD receptor complex. Physical alteration of the mersalyl-treated TCDD-receptor complex was evident from gel filtration, sucrose gradients, and DNA- and DEAE-Sepharose chromatography. Our results are in striking contrast to the effects of these reagents on steroid receptors, whose bound steroid hormone ligand is rapidly and reversibly displaced by lower concentrations of reagent. The observations are consistent (1) mersalyl inducing an irreversable change such as aggregation and / or denaturation of the unoccupiedAhreceptor as well as the receptor-ligand complex and (2) decreased sensitivity of the transformed TCDD-receptor complex compared to both the untransformed complex and the unoccupied receptor to disruption by these SH regeants (i.e., ligand binding and transformation elicit changes in the receptor which may modify the accessibility of SH groups).