Association of topoisomerase II with the hepatoma cell nuclear matrix: the role of intermolecular disulfide bond formation.
Association of topoisomerase II with the hepatoma cell nuclear matrix: the role of intermolecular disulfide bond formation.
复制标题
拓扑异构酶 II 与肝癌细胞核基质的关联：分子间二硫键形成的作用。
DOI：
10.1016/0014-4827(91)90071-2
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发表时间：
1991
影响因子：
3.7
通讯作者：
Shaper,JH
中科院分区：
文献类型：
--
作者：
Kaufmann,SH;Shaper,JH
先前的研究已经导致了关于核基质组分中的核酶拓扑异构酶（topo）II和topo I的回收的相互矛盾的数据。在本研究中，我们已经评估了系统地改变一个单一的提取过程中的HTC肝癌组织培养细胞的细胞核的亚分级这些酶的分布的影响。当在不可逆巯基封闭剂碘乙酰胺存在下分离核单层（通过在4 °C下用中性去污剂Nonidet-P40原位处理附着的细胞制备）时，随后用DNA酶I和RNA酶A处理，然后用1.6MNaCl处理，导致通过相差显微镜和常规透射电子显微镜评估的核内成分广泛耗尽的结构。这些结构含有原始核单层中存在的总蛋白质的12 ± 4%。与肌动蛋白和波形蛋白的分子量相当的层粘连蛋白和多肽是SDS-聚丙烯酰胺凝胶上存在的主要多肽。蛋白质印迹法显示，总核拓扑异构酶II分子的<5%存在于这些结构中。相反，当巯基交联剂连四硫酸钠（NaTT）取代碘乙酰胺，相同的提取程序产生的结构包含核仁和广泛的核内网络的组件。这些结构含有各种各样的非核纤层蛋白、非组蛋白核多肽，包括总核拓扑异构酶II的23 ± 4%。非还原条件下进行的SDS-聚丙烯酰胺凝胶电泳显示，拓扑异构酶II在这些核基质中是作为一个大的二硫键交联复合物的一部分。在1.6MNaCl中用还原剂处理这些结构释放拓扑异构体II。相比之下，topo I不形成二硫键交联的寡聚体，并且在4 °C下制备的这些核酸酶和盐抗性结构中的任何一个中都检测不到。为了评估体外热处理对拓扑异构酶分布的影响，在用核酸酶和1.6MNaCl处理之前，将核单层（在不存在碘乙酰胺和NaTT的情况下分离）加热至37 °C持续1小时。所得结构（保留总核蛋白的26 ± 5%）在形态上与NaTT稳定的核基质相似，并含有总核拓扑异构酶II的15 ± 4%。高分子量的二硫键交联寡聚体的拓扑异构酶II再次证明。在完整细胞中证明这些二硫键交联低聚物的尝试不成功。
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 cellsin situwith the neutral detergent Nonidet-P40 at 4 °C) were isolated in the presence of the irreversible sulfhydryl blocking reagent iodoacetamide, subsequent treatment with DNase I and RNase A followed by 1.6MNaCl 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 <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 crosslinked complex. Treatment of these structures with reducing agents in 1.6MNaCl 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 °C. To assess the effect ofin vitroheat treatment on the distribution of the topoisomerases, nuclear monolayers (isolated in the absence of iodoacetamide and NaTT) were heated to 37 °C for 1 h prior to treatment with nucleases and 1.6MNaCl. 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.