Bennett C F, Spector D L, Yeoman L C
J Cell Biol. 1986 Feb;102(2):600-9. doi: 10.1083/jcb.102.2.600.
A DNA-binding nonhistone protein, protein BA, was previously demonstrated to co-localize with U-snRNPs within discrete nuclear domains (Bennett, F. C., and L. C. Yeoman, 1985, Exp. Cell Res., 157:379-386). To further define the association of protein BA and U-snRNPs within these discrete nuclear domains, cells were fractionated in situ and the localization of the antigens determined by double-labeled immunofluorescence. Protein BA was extracted from the nucleus with the 2.0 M NaCl soluble chromatin fraction, while U-snRNPs were only partially extracted from the 2.0 M NaCl-resistant nuclear structures. U-snRNPs were extracted from the residual nuclear material by combined DNase I/RNase A digestions. Using an indirect immunoperoxidase technique and electron microscopy, protein BA was localized to interchromatinic regions of the cell nucleus. Protein BA was noted to share a number of chemical and physical properties with a family of cytoplasmic enzymes, the glutathione S-transferases. Comparison of the published amino acid composition of protein BA and glutathione S-transferases showed marked similarities. Nonhistone protein BA isolated from saline-EDTA nuclear extracts exhibited glutathione S-transferase activity with a variety of substrates. Substrate specificity and subunit analysis by SDS polyacrylamide gel electrophoresis revealed that it was a mixture of several glutathione S-transferase isoenzymes. Protein BA isolated from rat liver chromatin was shown by immunoblotting and peptide mapping techniques to be two glutathione S-transferase isoenzymes composed of the Yb and Yb' subunits. Glutathione S-transferase Yb subunits were demonstrated to be both nuclear and cytoplasmic proteins by indirect immunolocalization on rat liver cryosections. The identification of protein BA as glutathione S-transferase suggests that this family of multifunctional enzymes may play an important role in those nuclear domains containing U-snRNPs.
一种DNA结合非组蛋白BA蛋白，先前已被证明与U - snRNP在离散的核区域中共定位（贝内特，F.C.，和L.C.约曼，1985年，《实验细胞研究》，157:379 - 386）。为了进一步确定BA蛋白与U - snRNP在这些离散核区域内的关联，对细胞进行原位分级分离，并通过双标记免疫荧光确定抗原的定位。BA蛋白从细胞核中与2.0M NaCl可溶性染色质部分一起被提取出来，而U - snRNP仅从2.0M NaCl抗性核结构中部分被提取。U - snRNP通过DNA酶I/RNA酶A联合消化从残留核物质中被提取出来。使用间接免疫过氧化物酶技术和电子显微镜，BA蛋白被定位到细胞核的染色质间区域。注意到BA蛋白与一类细胞质酶——谷胱甘肽S - 转移酶具有许多化学和物理性质。已发表的BA蛋白和谷胱甘肽S - 转移酶的氨基酸组成比较显示出显著的相似性。从盐水 - EDTA核提取物中分离的非组蛋白BA蛋白对多种底物表现出谷胱甘肽S - 转移酶活性。通过SDS聚丙烯酰胺凝胶电泳进行的底物特异性和亚基分析表明，它是几种谷胱甘肽S - 转移酶同工酶的混合物。通过免疫印迹和肽图谱技术显示，从大鼠肝脏染色质中分离的BA蛋白是由Yb和Yb'亚基组成的两种谷胱甘肽S - 转移酶同工酶。通过对大鼠肝脏冷冻切片的间接免疫定位证明，谷胱甘肽S - 转移酶Yb亚基是核蛋白和细胞质蛋白。将BA蛋白鉴定为谷胱甘肽S - 转移酶表明，这一多功能酶家族可能在含有U - snRNP的那些核区域中起重要作用。
