Fields A P, Shaper J H
Department of Pharmacology and Molecular Sciences, Johns Hopkins University, School of Medicine, Baltimore, Maryland 21205.
J Cell Biol. 1988 Sep;107(3):833-40. doi: 10.1083/jcb.107.3.833.
We have isolated and partially characterized a major intranuclear matrix polypeptide from rat liver. This polypeptide, which is reversibly stabilized into the intranuclear matrix under conditions which promote intermolecular disulfide bond formation, has a Mr of 62,000 and pI of 6.8-7.2 as determined by two-dimensional IEF/SDS-PAGE. A chicken polyclonal antiserum was raised against the polypeptide purified from two-dimensional polyacrylamide gels. Affinity-purified anti-62-kD IgG was prepared and used to immunolocalize this polypeptide in rat liver tissue hepatocytes. In interphase hepatocytes the 62-kD antigen is localized in small, discrete patches within the nucleus consistent with the distribution of chromatin. The staining is most prominent at the nuclear periphery and somewhat less dense in the nuclear interior. Nucleoli and cytoplasm are devoid of staining. During mitosis the 62-kD antigen localizes to the condensed chromosomes with no apparent staining of cytoplasmic areas. The chromosomal staining during mitosis is uniform with no suggestion of the patching seen in interphase nuclei. Fractionation and immunoblotting studies using rat hepatoma tissue culture cells blocked in metaphase with colcemid confirm the chromosomal localization of this 62-kD intranuclear protein during mitosis. The 62-kD polypeptide fractionates completely with metaphase chromosome scaffolds generated by sequential treatment of isolated chromosomes with DNAse I and 1.6 M NaCl, suggesting that this major 62-kD intranuclear protein may be involved in maintaining metaphase chromosomal architecture.
我们从大鼠肝脏中分离出一种主要的核内基质多肽，并对其进行了部分特性分析。这种多肽在促进分子间二硫键形成的条件下可逆地稳定在核内基质中，通过二维IEF/SDS-PAGE测定，其分子量为62,000，等电点为6.8 - 7.2。用从二维聚丙烯酰胺凝胶中纯化的多肽制备了鸡多克隆抗血清。制备了亲和纯化的抗62-kD IgG，并用于在大鼠肝脏组织肝细胞中对该多肽进行免疫定位。在间期肝细胞中，62-kD抗原定位于核内与染色质分布一致的小的离散斑块中。染色在核周边最为明显，在核内部稍淡。核仁和细胞质无染色。在有丝分裂期间，62-kD抗原定位于浓缩染色体上，细胞质区域无明显染色。有丝分裂期间的染色体染色均匀，没有间期核中所见的斑块迹象。使用秋水仙酰胺阻断在中期的大鼠肝癌组织培养细胞进行分级分离和免疫印迹研究，证实了这种62-kD核内蛋白在有丝分裂期间的染色体定位。62-kD多肽与通过用DNA酶I和1.6 M NaCl顺序处理分离的染色体产生的中期染色体支架完全分离，表明这种主要的62-kD核内蛋白可能参与维持中期染色体结构。
