Feuerstein N, Ali I U
J Cell Biochem. 1985;29(3):253-63. doi: 10.1002/jcb.240290309.
The products of the ras gene family are related proteins at a molecular weight of 21 kDa, designated p21. In the present study we used two-dimensional gel electrophoresis to compare p21 proteins from five different normal and malignant cell lines. Using a known protein (3H-labeled translation initiation factor [eIF-4D]) as a standard internal marker for isoelectric point (pI), we show that p21 proteins from various cells differ only slightly in molecular weight (21-24 kDa) but express a wide variety in charge (pI 4.8 to 7) that could only be detected by the use of two-dimensional gel electrophoresis. p21 in NIH/3T3 cells was expressed as a single protein, which migrated at 21 kDa and pI 5.1. This peptide, which is probably the product of the normal cellular ras gene, was also detected in normal human lymphocytes. The synthesis of this peptide was not elevated in the transformed cells. However, transformation of NIH/3T3 fibroblasts and of human leukocytes was found to be associated with expression of qualitatively different forms of p21 peptides. Four additional p21-associated peptides of identical molecular weight (23 kDa), but multiple charge forms, were detected selectively in Kirsten murine sarcoma virus-transformed NIH/3T3 cells. Transformation of cells with Harvey murine sarcoma virus was found to be associated with prominent expression of two major pairs of p21-associated proteins, one at 21 kDa (pI, 5.2 and 5.3) and the other at 23 kDa (pI, 5.1 and 5.2). In HL-60 leukemic cells there was an additional, more acidic form (pI 5.0) of p21, which appeared to be absent or reduced in normal human lymphocytes. These results indicate that p21 from viral origin or cellular origin might be expressed in the cells in multiple charge forms. The capability to distinguish multiple forms of p21 and slight charge modifications associated with malignancy should call for the use of 2-D gel electrophoresis as an important tool in future studies involving p21 proteins.
ras基因家族的产物是分子量为21 kDa的相关蛋白，称为p21。在本研究中，我们使用二维凝胶电泳比较了来自五种不同正常和恶性细胞系的p21蛋白。使用一种已知蛋白（3H标记的翻译起始因子[eIF-4D]）作为等电点（pI）的标准内参，我们发现来自不同细胞的p21蛋白分子量仅略有差异（21 - 24 kDa），但电荷表现出广泛的多样性（pI 4.8至7），这只能通过二维凝胶电泳检测到。NIH/3T3细胞中的p21以单一蛋白形式表达，其迁移至21 kDa且pI为5.1。这种肽可能是正常细胞ras基因的产物，在正常人淋巴细胞中也能检测到。该肽的合成在转化细胞中并未升高。然而，发现NIH/3T3成纤维细胞和人白细胞的转化与性质不同的p21肽形式的表达有关。在 Kirsten 小鼠肉瘤病毒转化的 NIH/3T3 细胞中选择性地检测到另外四种分子量相同（23 kDa）但电荷形式多样的p21相关肽。发现用 Harvey 小鼠肉瘤病毒转化细胞与两对主要的p21相关蛋白的显著表达有关，一对在21 kDa（pI，5.2和5.3），另一对在23 kDa（pI，5.1和5.2）。在HL - 60白血病细胞中存在一种额外的、酸性更强的p21形式（pI 5.0），在正常人淋巴细胞中似乎不存在或减少。这些结果表明，病毒来源或细胞来源的p21可能在细胞中以多种电荷形式表达。区分多种形式的p21以及与恶性肿瘤相关的轻微电荷修饰的能力，应该促使二维凝胶电泳成为未来涉及p21蛋白研究的重要工具。
