ASSOCIATION OF METHIONYL-TRANSFER RNA-SYNTHETASE WITH DETERGENT-INSOLUBLE COMPONENTS OF THE ROUGH ENDOPLASMIC-RETICULUM
ASSOCIATION OF METHIONYL-TRANSFER RNA-SYNTHETASE WITH DETERGENT-INSOLUBLE COMPONENTS OF THE ROUGH ENDOPLASMIC-RETICULUM
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DOI：
10.1083/jcb.96.4.1138
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发表时间：
1983-01-01
影响因子：
7.8
通讯作者：
POLLARD, TD
中科院分区：
文献类型：
--
作者：
DANG, CV;YANG, DCH;POLLARD, TD
使用荧光抗体染色来确定甲硫氨酰-tRNA 合成酶与[大鼠袋鼠肾] PtK2 细胞中内质网的关联。 Triton X-100 提取后，70% 恢复的氨酰基-tRNA 合成酶活性存在于去污剂不溶部分中。该酶部分仍然定位于不溶性内质网抗原和核糖体，并用吖啶橙染色。通过荧光显微镜和电镜，发现洗涤剂不溶性残留物的组织取决于提取溶液的成分。用含有 EGTA [乙二醇双（β-氨基乙基醚）-N,N,N'',N''-四乙酸]、Triton X-100 和聚乙二醇的微管稳定缓冲液提取后，核糖体与膜残留物聚集成大簇。用含有 Triton X-100、蔗糖和 CaCl2 的缓冲液提取后，核糖体呈小簇状，并且几乎没有形态可识别的膜。在这两种情况下，甲硫氨酰-tRNA合成酶和一些内质网抗原在细胞中大致保持正常分布。双荧光染色显示甲硫氨酰-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没有形态学关联。这些观察结果表明，去污剂不溶性细胞成分（有时称为细胞骨架制剂）含有大量非丝状物质，包括核糖体和膜残留物。在推测如此复杂的细胞部分中的分子间关联时需要谨慎。
Fluorescent antibody staining was used to establish the association of methionyl-tRNA synthetase with the endoplasmic reticulum in [rat kangaroo kidney] 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 EM 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 [ethylene glycol bis(.beta.-aminoethyl ether)-N,N,N'',N''-tetraacetic acid], Triton X-100, and polyethylene glycol, the ribosomes were aggregated in large clusters with remnants of membranes. After extraction with a buffer containing Triton X-100, sucrose, and CaCl2, 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.