Association of methionyl-tRNA synthetase with detergent-insoluble components of the rough endoplasmic reticulum-猫眼课题宝

甲硫氨酰-tRNA合成酶与粗面内质网洗涤剂不溶性成分的关联

DOI：

发表时间：

1983

期刊：

Journal of Cell Biology

影响因子：

7.8

通讯作者：

中科院分区：

生物学1区

文献类型：

作者：

C. Dang;David C. H. Yang;T. Pollard

通过荧光抗体染色，我们在PtK2细胞中建立了甲硫酰-tRNA合成酶与内质网的联系。经Triton X-100提取后，回收的氨基酰tRNA合成酶活性的70%存在于洗涤剂不溶部分。酶的这一部分仍然与不溶性内质网抗原和核糖体结合，这些核糖体被丫啶橙染色。荧光显微镜和电子显微镜都发现，洗涤剂不溶残留物的组织取决于提取液的组成。在用含有EGTA、Triton X-100和聚乙二醇的微管稳定缓冲液(Osburn，M.和K.Weber，1977，Cell，12：561-571)提取后，核糖体聚集在带有膜残留物的大簇中。用含有Triton X-100、蔗糖和CaCl2(Fulton，A.B.，K.M.Wang，and S.Penman，1980，Cell，20：849-857)的缓冲液提取核糖体后，核糖体呈小簇状，几乎没有形态可识别的膜。在这两种情况下，甲硫酰-tRNA合成酶和一些内质网抗原在细胞内保持大致正态分布。双重荧光染色显示，甲硫酰-tRNA合成酶与PtK2细胞的微管、肌动蛋白或细胞角蛋白纤维系统在形态上没有关联。这些观察表明，不溶于洗涤剂的细胞成分，有时被称为“细胞骨架”制剂，含有大量的非丝状物质，包括核糖体和膜残留物。在推测如此复杂的细胞组分中的分子间相互作用时需要谨慎。

Using fluorescent antibody staining, we have established the association of methionyl-tRNA synthetase with the endoplasmic reticulum in PtK2 cells. After Triton X-100 extraction, 70% of the recovered aminoacyl-tRNA synthetase activity was found in the detergent-insoluble fraction. This fraction of the enzyme remained localized with insoluble endoplasmic reticulum antigens and with ribosomes, which were stained with acridine orange. By both fluorescence microscopy and electron microscopy the organization of the detergent-insoluble residue was found to depend on the composition of the extracting solution. After extraction with a microtubule-stabilizing buffer containing EGTA, Triton X-100, and polyethylene glycol (Osburn, M., and K. Weber, 1977, Cell, 12:561-571) the ribosomes were aggregated in large clusters with remnants of membranes. After extraction with a buffer containing Triton X-100, sucrose, and CaCl2 (Fulton, A. B., K. M. Wang, and S. Penman, 1980, Cell, 20:849-857), the ribosomes were in small clusters and there were few morphologically recognizable membranes. In both cases the methionyl-tRNA synthetase and some endoplasmic reticulum antigens retained approximately their normal distribution in the cell. Double fluorochrome staining showed no morphological association of methionyl- tRNA synthetase with the microtubule, actin, or cytokeratin fiber systems of PtK2 cells. These observations demonstrate that detergent- insoluble cellular components, sometimes referred to as "cytoskeletal" preparations, contain significant amounts of nonfilamentous material including ribosomes, and membrane residue. Caution is required in speculating about intermolecular associations in such a complex cell fraction.

Stoichiometry and composition of an aminoacyl-tRNA synthetase complex from rat liver.