Kaufmann S H, Shaper J H
Department of Pharmacology, Johns Hopkins University School of Medicine, Johns Hopkins Hospital, Baltimore, Maryland 21205.
Exp Cell Res. 1991 Feb;192(2):511-23. doi: 10.1016/0014-4827(91)90071-2.
Previous studies have resulted in conflicting data regarding the recovery of the nuclear enzymes topoisomerase (topo) II and topo I in the nuclear matrix fraction. In the present study we have assessed the effect of systematically altering a single extraction procedure on the distribution of these enzymes during the subfractionation of nuclei from HTC hepatoma tissue culture cells. When nuclear monolayers (prepared by treating attached cells in situ with the neutral detergent Nonidet-P40 at 4 degrees C) were isolated in the presence of the irreversible sulfhydryl blocking reagent iodoacetamide, subsequent treatment with DNase I and RNase A followed by 1.6 M NaCl resulted in structures which were extensively depleted of intranuclear components as assessed by phase contrast microscopy and conventional transmission electron microscopy. These structures contained 12 +/- 4% of the total protein present in the original nuclear monolayers. The lamins and polypeptides with molecular weights comparable to those of actin and vimentin were the predominant polypeptides present on SDS-polyacrylamide gels. Western blotting revealed that less than 5% of the total nuclear topo II molecules were present in these structures. In contrast, when the sulfhydryl cross-linking reagent sodium tetrathionate (NaTT) was substituted for iodoacetamide, the same extraction procedure yielded structures containing components of the nucleolus and an extensive intranuclear network. These structures contained a wide variety of nonlamin, nonhistone nuclear polypeptides including 23 +/- 4% of the total nuclear topo II. SDS-polyacrylamide gel electrophoresis performed under nonreducing conditions revealed that topo II in these nuclear matrices was present as part of a large disulfide cross-linked complex. Treatment of these structures with reducing agents in 1.6 M NaCl released the topo II. In contrast, topo I did not form disulfide cross-linked oligomers and was not detectable in any of these nuclease- and salt-resistant structures prepared at 4 degrees C. To assess the effect of in vitro heat treatment on the distribution of the topoisomerases, nuclear monolayers (isolated in the absence of iodoacetamide and NaTT) were heated to 37 degrees C for 1 h prior to treatment with nucleases and 1.6 M NaCl. The resulting structures (which retained 26 +/- 5% of the total nuclear protein) were morphologically similar to the NaTT-stabilized nuclear matrices and contained 15 +/- 4% of the total nuclear topo II. High-molecular-weight disulfide cross-linked oligomers of topo II were again demonstrated. Attempts to demonstrate these disulfide cross-linked oligomers in intact cells were unsuccessful.
以往的研究在核基质组分中核酶拓扑异构酶（topo）II和topo I的恢复方面得出了相互矛盾的数据。在本研究中，我们评估了系统改变单一提取程序对HTC肝癌组织培养细胞核分级分离过程中这些酶分布的影响。当在不可逆的巯基阻断剂碘乙酰胺存在下分离核单层（通过在4℃用中性去污剂Nonidet-P40原位处理贴壁细胞制备）时，随后用DNase I和RNase A处理，再用1.6 M NaCl处理，通过相差显微镜和传统透射电子显微镜评估，得到的结构中核内成分大量减少。这些结构含有原始核单层中总蛋白的12±4%。在SDS-聚丙烯酰胺凝胶上，核纤层蛋白以及分子量与肌动蛋白和波形蛋白相当的多肽是主要的多肽。蛋白质印迹分析表明，这些结构中存在的核topo II分子不到总核topo II分子的5%。相反，当用巯基交联剂连四硫酸钠（NaTT）替代碘乙酰胺时，相同的提取程序产生的结构包含核仁成分和广泛的核内网络。这些结构含有多种非核纤层、非组蛋白核多肽，包括总核topo II的23±4%。在非还原条件下进行的SDS-聚丙烯酰胺凝胶电泳表明，这些核基质中的topo II以大的二硫键交联复合物的一部分形式存在。在1.6 M NaCl中用还原剂处理这些结构会释放出topo II。相反，topo I不形成二硫键交联的寡聚体，并且在4℃制备的任何这些耐核酸酶和耐盐结构中都检测不到。为了评估体外热处理对拓扑异构酶分布的影响，在没有碘乙酰胺和NaTT的情况下分离的核单层在用核酸酶和1.6 M NaCl处理之前，加热至37℃1小时。得到的结构（保留了总核蛋白的26±5%）在形态上与NaTT稳定的核基质相似，并且含有总核topo II的15±4%。再次证明了topo II的高分子量二硫键交联寡聚体。在完整细胞中证明这些二硫键交联寡聚体的尝试未成功。
