Vinores S A, Churey J J, Haller J M, Schnabel S J, Custer R P, Sorof S
Proc Natl Acad Sci U S A. 1984 Apr;81(7):2092-6. doi: 10.1073/pnas.81.7.2092.
A 14,000-dalton polypeptide was previously reported to be the principal protein target of the carcinogen N-2-fluorenylacetamide (2-acetylaminofluorene) in liver cytosol at the start of hepatocarcinogenesis in rats. The 14,000-dalton polypeptide was purified to homogeneity according to gel electrophoreses in both NaDodSO4-containing medium and acetic acid/urea and also by immunogenicity. An immunologically related form of the cytosolic target polypeptide has now been found to be present in the nuclei of normal rat liver as a 17,500-dalton polypeptide that is firmly and ionically bound to chromatin. Serial salt extractions of isolated liver nuclei or chromatin at 0.15 and 0.35 ionic strengths fail to dissolve the bound polypeptide, according to electrophoretic transfer immunoblot analyses. Most of the 17,500-dalton polypeptide is extracted at 0.65 ionic strength, the remainder at 1.2, and none at 2.0, nor thereafter in 8 M urea. In addition, short-term digestion of purified liver nuclei with micrococcal nuclease solubilizes the 17,500-dalton polypeptide. All three protocols also solubilize low levels of intermediate 17,500- to 14,000-dalton species, the latter size being the same as that of the cytosolic protein target of the carcinogen. The presence of protease inhibitors during the isolations and extractions of the nuclei and chromatin reduces the amounts of these smaller polypeptides. In normal rat liver only nuclei and cytoplasm of hepatocytes contain reactive antigen according to peroxidase-antiperoxidase immunohistochemistry, staining most intensely perilobularly, less in the lobular midzone, and least centrilobularly. The nuclei of the perilobular hepatocytes constitute the strongest staining compartment within all of normal liver. Of 22 nonhepatic tissues of normal rats, 16 contain relatively few cells with immunoreactive cytoplasm. Nonhepatic nuclear antigen is present only in villar crest cells of duodenum (which are normally exposed to liver bile), also having cytoplasmic antigen as well. Five kinds of evidence appear to connect the chromatin-bound 17,500-dalton polypeptide of normal liver nuclei to the cytosolic 14,000-dalton polypeptide that is the principal target of the carcinogen early during hepatocarcinogenesis in rats. The present findings indicate a direct connection between a chromosomal protein and the immediate principal cytosolic protein target of a carcinogen.
先前有报道称，在大鼠肝癌发生初期，一种14000道尔顿的多肽是致癌物N - 2 - 芴基乙酰胺（2 - 乙酰氨基芴）在肝细胞质中的主要蛋白质靶点。通过在含十二烷基硫酸钠的介质以及乙酸/尿素中进行凝胶电泳，并依据免疫原性，将这种14000道尔顿的多肽纯化至同质状态。现已发现，在正常大鼠肝脏的细胞核中存在一种与胞质靶点多肽具有免疫相关性的形式，它是一种17500道尔顿的多肽，牢固地以离子键形式与染色质结合。根据电泳转移免疫印迹分析，在0.15和0.35离子强度下对分离的肝细胞核或染色质进行连续盐提取，无法溶解结合的多肽。大部分17500道尔顿的多肽在0.65离子强度下被提取出来，其余的在1.2离子强度下被提取，在2.0离子强度下以及之后在8M尿素中均无提取出来的。此外，用微球菌核酸酶对纯化的肝细胞核进行短期消化可溶解17500道尔顿的多肽。这三种方法还能溶解少量分子量介于17500至14000道尔顿之间的中间产物，后者的大小与致癌物的胞质蛋白靶点相同。在细胞核和染色质的分离及提取过程中加入蛋白酶抑制剂，可减少这些较小多肽的量。根据过氧化物酶 - 抗过氧化物酶免疫组织化学方法，在正常大鼠肝脏中，只有肝细胞的细胞核和细胞质含有反应性抗原，小叶周边染色最强，小叶中区较弱，小叶中央区最弱。小叶周边肝细胞的细胞核是整个正常肝脏中染色最强的区域。在正常大鼠的22种非肝脏组织中，16种含有相对较少的具有免疫反应性细胞质的细胞。非肝脏细胞核抗原仅存在于十二指肠的绒毛嵴细胞中（这些细胞通常暴露于肝胆汁中），其细胞质中也有抗原。五种证据似乎将正常肝细胞核中与染色质结合的17500道尔顿多肽与大鼠肝癌发生早期作为致癌物主要靶点的14000道尔顿胞质多肽联系起来。目前的研究结果表明了一种染色体蛋白与致癌物直接的主要胞质蛋白靶点之间的直接联系。
