Gerner C, Holzmann K, Grimm R, Sauermann G
Institute of Tumor Biology-Cancer Research, University of Vienna, Austria.
J Cell Biochem. 1998 Dec 1;71(3):363-74. doi: 10.1002/(sici)1097-4644(19981201)71:3<363::aid-jcb5>3.0.co;2-w.
Comparative analysis of nuclear matrix proteins by two-dimensional electrophoresis may be greatly impaired by copurifying cytoskeletal proteins. The present data show that the bulk of adhering cytofilaments may mechanically be removed by shearing of nuclei pretreated with vanadyl ribonucleoside complexes. Potential mechanisms of action not based on ribonuclease inhibition are discussed. To individually preserve the integrity of nuclear structures, we developed protocols for the preparation of nuclear matrices from three categories of cells, namely leukocytes, cultured cells, and tissue cells. As exemplified with material from human lymphocytes, cultured amniotic cells, and liver tissue cells, the resulting patterns of nuclear matrix proteins appeared quite similar. Approximately 300 spots were shared among the cell types. Forty-nine of these were identified, 21 comprising heterogeneous nuclear ribonucleoproteins. Heterogeneous nuclear ribonucleoproteins L and nuclear lamin B2 isoforms were identified by amino acid sequencing and mass spectrometry. However, individually expressed proteins, such as the proliferating cell nuclear antigen, also pertained following application of the protocols. Thus, enhanced resolution and comparability of proteins improve systematic analyses of nuclear matrix proteins from various cellular sources.
通过二维电泳对核基质蛋白进行比较分析可能会因共纯化细胞骨架蛋白而受到极大影响。目前的数据表明，大部分附着的细胞丝可通过对用钒核糖核苷复合物预处理的细胞核进行剪切而机械去除。讨论了不基于核糖核酸酶抑制作用的潜在作用机制。为了分别保持核结构的完整性，我们开发了从三类细胞（即白细胞、培养细胞和组织细胞）制备核基质的方案。以人淋巴细胞、培养的羊膜细胞和肝组织细胞的材料为例，所得核基质蛋白的模式看起来非常相似。细胞类型之间共有约300个斑点。其中49个被鉴定出来，21个包含不均一核核糖核蛋白。通过氨基酸测序和质谱鉴定了不均一核核糖核蛋白L和核纤层蛋白B2亚型。然而，在应用这些方案后，诸如增殖细胞核抗原等个别表达蛋白也存在。因此，蛋白质分辨率和可比性提高，有助于对来自各种细胞来源的核基质蛋白进行系统分析。
