Isoform composition and stoichiometry of the approximately 90-kDa heat shock protein associated with glucocorticoid receptors.
Isoform composition and stoichiometry of the approximately 90-kDa heat shock protein associated with glucocorticoid receptors.
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DOI：
10.1016/s0021-9258(18)68698-3
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发表时间：
1988-05
期刊：
影响因子：
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通讯作者：
D. Mendel;E. Ortí
中科院分区：
文献类型：
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作者：
D. Mendel;E. Ortí
我们观察到，从 WEHI-7 小鼠胸腺瘤细胞纯化的非活化糖皮质激素受体的约 90 kDa 非类固醇结合成分（已被鉴定为约 90 kDa 热休克蛋白）在变性和还原条件下的聚丙烯酰胺凝胶电泳过程中始终作为双峰迁移。最近报道，鼠Meth A细胞含有肿瘤特异性移植抗原(TSTA)，其与约90-kDa热休克蛋白相关或相同(Ullrich, S.J.、Robinson, E.A.、Law, L.W.、Willingham, M.和Appella, E.(1986) Proc. Natl. Acad. Sci. U.S.A. 83， 3121-3125）。 TSTA 和从这些细胞中分离出的大约 90 kDa 的热休克蛋白作为两种具有相似分子质量和电荷的同工型存在的观察结果表明，我们观察到的双联体也是由于两种同工型的存在所致。然而，与 TSTA 不同的是，TSTA 似乎含有相似相对丰度的两种同工型，未活化的糖皮质激素受体复合物似乎主要含有较低分子量的同工型。因此，我们进行了这项研究，以确定 TSTA 和糖皮质激素受体的约 90 kDa 成分是否确实相关，以确定受体是否优先结合约 90 kDa 热休克蛋白的一种亚型，并研究非激活受体复合物的化学计量。通过比较 Meth A TSTA 和受体的约 90 kDa 成分与 AC88 单克隆抗体（对约 90 kDa 热休克蛋白具有特异性）和针对 Meth A TSTA 的多克隆抗体的反应，我们发现这两种蛋白无法区分，而且可能相同。然后，我们使用 BuGR1（针对糖皮质激素受体的类固醇结合亚基）和 AC88 单克隆抗体分别从使用 [35S] 蛋氨酸生长 48 小时的 WEHI-7 细胞中纯化受体相关的和游离的约 90 kDa 热休克蛋白，以代谢标记蛋白质至稳态。在变性和还原条件下通过聚丙烯酰胺凝胶电泳分析蛋白质后，根据 35S 计数和每种异构体的已知蛋氨酸含量确定每个样品中两种异构体的相对量。我们发现大约四分之三的受体相关蛋白和游离的约 90 kDa 热休克蛋白以较低分子量的异构体形式存在，表明任一异构体在受体中都没有优先结合。长期代谢标记方法也使我们能够指导
We have observed that the approximately 90-kDa non-steroid-binding component of nonactivated glucocorticoid receptors purified from WEHI-7 mouse thymoma cells (which has been identified as the approximately 90-kDa heat shock protein) consistently migrates as a doublet during polyacrylamide gel electrophoresis under denaturing and reducing conditions. It has recently been reported that murine Meth A cells contain a tumor-specific transplantation antigen (TSTA) which is related or identical to the approximately 90-kDa heat shock protein (Ullrich, S.J., Robinson, E.A., Law, L.W., Willingham, M., and Appella, E. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 3121-3125). The observation that TSTA and the approximately 90-kDa heat shock protein isolated from these cells exists as two isoforms of similar molecular mass and charge has suggested to us that the doublet we observed is also due to the existence of two isoforms. However, unlike TSTA, which appears to contain the two isoforms in similar relative abundance, nonactivated glucocorticoid-receptor complexes seem to contain predominantly the lower molecular mass isoform. We have therefore conducted this study to determine whether TSTA and the approximately 90-kDa component of glucocorticoid receptors are indeed related, to establish whether the receptor preferentially binds one isoform of the approximately 90-kDa heat shock protein, and to investigate the stoichiometry of the nonactivated receptor complex. By comparing Meth A TSTA and the approximately 90-kDa component of the receptor in their reactions with the AC88 monoclonal antibody (specific for the approximately 90-kDa heat shock protein) and a polyclonal antibody directed against Meth A TSTA, we found that these two proteins are indistinguishable and probably identical. We then used the BuGR1 (directed against the steroid-binding subunit of glucocorticoid receptors) and AC88 monoclonal antibodies to purify, respectively, receptor-associated and free approximately 90-kDa heat shock protein from WEHI-7 cells grown for 48 h with [35S]methionine to metabolically label proteins to steady state. Following analysis of the proteins by polyacrylamide gel electrophoresis under denaturing and reducing conditions, the relative amounts of the two isoforms in each sample were determined from the 35S counts and the known methionine content of each isoform. We found that approximately three-quarters of both the receptor-associated and the free approximately 90-kDa heat shock protein is present as the lower molecular weight isoform, indicating no preferential binding of either isoform in the receptor. The long-term metabolic labeling approach has also enabled us to direc