ALTERATIONS IN NUCLEAR ANATOMY BY CHEMICAL MODIFICATION OF PROTEINS IN ISOLATED RAT-LIVER NUCLEI
ALTERATIONS IN NUCLEAR ANATOMY BY CHEMICAL MODIFICATION OF PROTEINS IN ISOLATED RAT-LIVER NUCLEI
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DOI：
10.1016/0014-4827(84)90704-3
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发表时间：
1984-01-01
影响因子：
3.7
通讯作者：
SCHINDLER, M
中科院分区：
文献类型：
--
作者：
SCHINDLER, M
用针对伯胺的试剂柠檬酸酐处理大鼠全肝核。核的形态和光散射特性发生了巨大的变化。对DNA和RNA含量的分析表明，DNA从细胞核中释放的半衰期很短，约为。2-4秒显示双相释放剖面。RNA同样被释放，但具有单相特征。对修饰核的SDS-PAGE（十二烷基硫酸钠-聚丙烯酰胺凝胶电泳）凝胶的分析表明，随着修饰程度的增加，核基质（层蛋白）多肽逐渐富集。H1组蛋白随修饰试剂浓度的变化而定量丢失，而。apprx。50%的核小体组蛋白与不含DNA和rna的细胞核共沉积。在2 mM EGTA存在下的修饰释放了所有的DNA和RNA。剩余1%)，同时保留了核基质、核仁和核糖核蛋白的结构特征（主要是hn[异质核]RNA组A和B）。这些缺乏核酸的结构被称为核化石，以区别于高盐洗涤剂制备的空核袋、核残余物或核支架。在2% Triton X-100的存在下进行修饰，其结构类似于核化石（EGTA[乙二醇双β]）。-氨基乙醚)N，N，N‘，N’ -四乙酸]处理)，但缺少双双分子层和51K[千道尔顿]多肽，这是其他结构的主要组成部分。原子核的化学修饰为研究离子相互作用在控制核结构中的作用提供了一种实验方法。因此，Citraconylation可能具有两种功能：作为细胞核的蛋白质特异性扰动，能够简单而快速地制备一系列结构变体，用于核相互作用的分析；为控制核酸-多肽相互作用提供了一个基于蛋白质电荷翻译后改变的范例。
Whole rat liver nuclei were treated with citraconic anhydride, a reagent specific for primary amines. Dramatic changes were observed in nuclear morphology and light scattering properties. An analysis for DNA and RNA content suggested that DNA was released from the nuclei with a short half-time, .apprx. 2-4 s demonstrating a biphasic release profile. RNA was similarly released but with a monophasic profile. Analysis of SDS-PAGE[sodium dodecyl sulfate-polyacrylamide gel electrophoresis] gels of modified nuclei demonstrated a progressive enrichment of nuclear matrix (lamins) polypeptides with extent of modification. H1 histone was quantitatively lost as a function of modification reagent concentration, while .apprx. 50% of the nucleosomal histones cosedimented with DNA- and RNA-free nuclei. Modification in the presence of 2 mM EGTA released all the DNA and RNA ((.ltoreq. 1% remaining) while retaining structures characteristic of nuclear matrix, nucleoli and ribonucleoprotein (predominantly hn[heterogeneous nuclear]RNA group A and B). These nucleic acid-deficient structures were termed nuclear fossils to differentiate them from high salt detergent-prepared empty nuclear sacks, nuclear remnants or nuclear scaffolds. Modification in the presence of 2% Triton X-100 results in structures similar to the nuclear fossils (EGTA [ethylene glycol bis(.beta.-aminoethyl ether) N,N,N'',N''-tetracetic acid] treatment), but missing the double bilayer and a 51K [kilodalton] polypeptide that is a major component of the other structures. The use of chemical modification of the nucleus provides an experimental approach for examining the role of ionic interactions in controlling nuclear structure. Citraconylation may thus serve 2 functions: as a protein-specific perturbant of nuclei capable of simply and rapidly preparing a range of structural variants for the analysis of nuclear interactions; offer a paradigm for control of nucleic acid-polypeptide interactions based on post-translational alterations in protein charge.