A SUBSET OF NON-HISTONE NUCLEAR PROTEINS REVERSIBLY STABILIZED BY THE SULFHYDRYL CROSS-LINKING REAGENT TETRATHIONATE - POLYPEPTIDES OF THE INTERNAL NUCLEAR MATRIX
A SUBSET OF NON-HISTONE NUCLEAR PROTEINS REVERSIBLY STABILIZED BY THE SULFHYDRYL CROSS-LINKING REAGENT TETRATHIONATE - POLYPEPTIDES OF THE INTERNAL NUCLEAR MATRIX
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DOI：
10.1016/0014-4827(84)90208-8
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发表时间：
1984-01-01
影响因子：
3.7
通讯作者：
SHAPER, JH
中科院分区：
文献类型：
--
作者：
KAUFMANN, SH;SHAPER, JH
当大鼠肝细胞核中分离的存在下的不可逆的巯基阻断剂碘乙酰胺，消化与DNA酶I和RNA酶A，并提取与1.6 M NaCl，核被膜（NE）球耗尽核内材料，如通过薄切片电子显微镜分析，获得。双向等电聚焦（IEF）/SDS-PAGE [十二烷基硫酸钠，聚丙烯酰胺凝胶电泳]和非平衡pH梯度电泳（NEPHGE）/SDS-PAGE显示，主要的多肽是核纤层蛋白A、B和C。在没有巯基封闭剂的情况下分离的细胞核产生盐和核酸酶抗性结构，其中含有稀疏但可证明的核内物质。除了核纤层蛋白之外，还发现了许多非组蛋白多肽。用巯基交联剂连四硫酸钠（NaTT）处理的核在暴露于核酸酶和1.6 M NaCl后产生核基质样结构，该结构含有广泛的核内网络和除NE外的核仁组分。增加量的非核纤层蛋白、非组蛋白多肽与这些结构一起回收。随后处理这些NaTT-交联的结构与还原剂在1.0 M NaCl选择性地溶解的核内成分，但留下的核膜明显完整。核纤层蛋白保持可沉积性，并且几乎不存在于可溶性（核内）物质中。相反，主要溶解的多肽是分别在核纤层蛋白B和C附近迁移的68和63 kD [千道尔顿]多肽，但通过免疫印迹和一维肽图谱可与核纤层蛋白区分开;一系列碱性60- 70 kD [千道尔顿]多肽（pI > 8.0），其不被抗核纤层蛋白抗血清识别;酸性（pI 5.3）38 kD多肽;和许多高分子量（> 100 kD）多肽。这些观察结果不仅提示了将大鼠肝细胞核的基质结构分级分离成生化和形态学离散组分的方便方法，而且还鉴定了主要非核纤层蛋白、非组蛋白核多肽的亚组（包括20%的总核蛋白），其分子间相互作用可以通过NaTT形成的分子间二硫键而明显可逆地稳定。
When rat liver nuclei are isolated in the presence of the irreversible sulfhydryl-blocking reagent iodoacetamide, digested with DNase I and RNase A, and extracted with 1.6 M NaCl, nuclear envelope (NE) spheres depleted of intranuclear material, as analyzed by thin-section electron microscopy, are obtained. Two-dimensional isoelectric focusing (IEF)/SDS-PAGE [sodium dodecylsulfate, polyacrylamide gel electrophoresis] and non-equilibrium pH gradient electrophoresis (NEPHGE)/SDS-PAGE reveal that the predominant polypeptides are lamins A, B and C. Nuclei isolated in the absence of sulfhydryl blocking reagents yield salt- and nuclease-resistant structures which contain sparse but demonstrable intranuclear material. A number of non-histone polypeptides are seen in addition to the lamins. Nuclei treated with the sulfhydryl cross-linking reagent sodium tetrathionate (NaTT) yield, after exposure to nucleases and 1.6 M NaCl, nuclear matrix-like structures containing an extensive intranuclear network and components of the nucleolus in addition to the NE. Increased amounts of the non-lamin, non-histone polypeptides are recovered with these structures. Subsequent treatment of these NaTT-cross-linked structures with reducing agents in 1.0 M NaCl selectively solubilizes the intranuclear components but leaves the nuclear envelope apparently intact. The lamins remain sedimentable and are virtually absent from the soluble (intranuclear) material. Instead, the major solubilized polypeptides are 68 and 63 kD [kilodalton] polypeptides which migrate in the vicinity of lamins B and C, respectively, but are distinguishable from the lamins by immunoblotting and by uni-dimensional peptide mapping; a series basic 60-70kd [kilodalton] polypeptides (pI > 8.0) which are not recognized by anti-lamin antisera; an acidic (pI 5.3) 38 kD polypeptide; and a number of high molecular mass (> 100 kD) polypeptides. These observations not only suggest a convenient method for fractionating matrix structures from rat liver nuclei into biochemically and morphologically discrete components, but also identify a subset of major non-lamin, non-histone nuclear polypeptides (comprising .apprx. 20% of the total nuclear protein) whose intermolecular interactions can be reversibly stabilized apparently by intermolecular disulfide bond formation by NaTT.