Simons S S
J Biol Chem. 1987 Jul 15;262(20):9669-75.
The specificity of protein labeling by an affinity label of glucocorticoid receptors, dexamethasone 21-mesylate (Dex-Mes), was investigated using bovine serum albumin (BSA) as a model. During the early stages of [3H]Dex-Mes labeling at pH 8.8, approximately 90% of the covalent bond formation occurred at the one non-oxidized cysteine (Cys-34) of BSA. The nonspecific labeling was equally distributed over the rest of the BSA molecule. [3H]Dex-Mes labeling of Cys-34 was totally, and specifically inhibited by nearly stoichiometric amounts of the thiol-specific reagent methyl methanethiolsulfonate (MMTS). Thus both Dex-Mes and MMTS appear to react very selectively with thiols under our conditions. In reactions with hepatoma tissue culture (HTC) cell glucocorticoid receptors, MMTS was equally efficient in preventing [3H]dexamethasone binding to receptors and [3H]Dex-Mes labeling of the 98-kDa receptor protein. These results indicate that Dex-Mes labeling of the glucocorticoid receptor involves covalent reaction with at least one cysteine in the steroid binding site of the receptor. Small (approximately 1600-dalton) fragments of the [3H]Dex-Mes-labeled 98-kDa receptor were generated by limit proteolysis with trypsin, chymotrypsin, and Staphylococcus aureus V8 protease under denaturing conditions. Data from these fragments on 15% sodium dodecyl sulfate-polyacrylamide gels were consistent with all of the covalent [3H] Dex-Mes being located on one or a few cysteines in one approximately 15-residue stretch of the receptor. Further studies revealed no differences in the limit protease digestion patterns of activated and unactivated [3H]Dex-Mes-labeled receptors with trypsin, chymotrypsin, or V8 protease under denaturing conditions. These data suggest that activation does not cause any major covalent modifications of the amino acids immediately surrounding the affinity-labeled cysteine(s) of the steroid binding site.
以牛血清白蛋白（BSA）为模型，研究了糖皮质激素受体亲和标记物甲磺酸地塞米松21酯（Dex-Mes）对蛋白质标记的特异性。在pH 8.8条件下进行[3H]Dex-Mes标记的早期阶段，约90%的共价键形成发生在BSA的一个未氧化半胱氨酸（Cys-34）处。非特异性标记均匀分布在BSA分子的其余部分。Cys-34的[3H]Dex-Mes标记完全被近乎化学计量的硫醇特异性试剂甲硫醇磺酸甲酯（MMTS）特异性抑制。因此，在我们的条件下，Dex-Mes和MMTS似乎都能与硫醇非常选择性地反应。在与肝癌组织培养（HTC）细胞糖皮质激素受体的反应中，MMTS在阻止[3H]地塞米松与受体结合以及[3H]Dex-Mes对98-kDa受体蛋白的标记方面同样有效。这些结果表明，糖皮质激素受体的Dex-Mes标记涉及与受体类固醇结合位点中至少一个半胱氨酸的共价反应。在变性条件下，用胰蛋白酶、糜蛋白酶和金黄色葡萄球菌V8蛋白酶进行有限度的蛋白水解，产生了[3H]Dex-Mes标记的98-kDa受体的小片段（约1600道尔顿）。在15%十二烷基硫酸钠-聚丙烯酰胺凝胶上这些片段的数据与所有共价[3H] Dex-Mes位于受体一个约15个残基片段中的一个或几个半胱氨酸上一致。进一步的研究表明，在变性条件下，活化和未活化的[3H]Dex-Mes标记受体与胰蛋白酶、糜蛋白酶或V8蛋白酶的有限度蛋白酶消化模式没有差异。这些数据表明，活化不会导致类固醇结合位点亲和标记半胱氨酸周围的氨基酸发生任何主要的共价修饰。
