Biochemical and molecular analysis of the mammalian cytoplasmic dynein intermediate chain
Biochemical and molecular analysis of the mammalian cytoplasmic dynein intermediate chain
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DOI：
10.1006/meth.2000.1083
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发表时间：
2000-12-01
期刊：
影响因子：
4.8
通讯作者：
Pfister, KK
中科院分区：
文献类型：
--
作者：
Brill, LB;Pfister, KK
细胞质动力蛋白是一种多亚基蛋白复合物，负责膜性细胞器和其他货物沿沿着的细胞内运动。重链包含马达结构域，而中间链和其他亚基对于结合货物是重要的。至少有五种不同的中间链多肽，它们是两个基因选择性剪接的产物。细胞质动力蛋白中间链也被磷酸化。不同中间链mRNA的表达通过逆转录-聚合酶链反应表征，所述逆转录-聚合酶链反应使用适合于可变剪接位点的寡核苷酸引物。通过细胞质动力蛋白样品的二维凝胶分析确定不同中间链多肽同种型的存在。通过用蛋白磷酸酶处理免疫沉淀的细胞质动力蛋白并分析二维凝胶电泳后多肽斑点分布的变化来确定多肽的磷酸化状态。(C)北京大学出版社.
Cytoplasmic dynein is a multisubunit protein complex responsible for the intracellular movement of membranous organelles and other cargo along microtubules. The heavy chains contain the motor domains, while the intermediate chain and other subunits are important for binding to cargo. There are at least five different intermediate chain polypeptides, the products of alternative splicing of two genes. The cytoplasmic dynein intermediate chains are also phosphorylated. The expression of the different intermediate chain mRNAs is characterized by reverse transcription-polymerase chain reactions using oligonucleotide primers appropriate for the alternative splicing sites. The presence of the different intermediate chain polypeptide isoforms is determined by two-dimensional gel analysis of cytoplasmic dynein samples. The phosphorylation state of the polypeptides is determined by treatment of immunoprecipitated cytoplasmic dynein with protein phosphatase and analysis of changes in polypeptide spot distribution after two-dimensional gel electrophoresis. (C) 2000 Academic Press.