Solubilization of SV40 plasma-membrane-associated large tumor antigen using single-phase concentrations of 1-butanol.
Solubilization of SV40 plasma-membrane-associated large tumor antigen using single-phase concentrations of 1-butanol.
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使用单相浓度的 1-丁醇溶解 SV40 质膜相关的大肿瘤抗原。
DOI：
10.1002/mc.2940020607
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发表时间：
1989
影响因子：
4.6
通讯作者：
Butel,JS
中科院分区：
文献类型：
--
作者：
Maxwell,SA;Santos,M;Wong,C;Rasmussen,G;Butel,JS
猴病毒40（SV 40）转化蛋白（大肿瘤抗原（T-ag））与转化细胞质膜相互作用的性质尚不清楚。我们在此报告，SV 40质膜相关大肿瘤抗原（pmT-ag）可以通过使用单相浓度的1-丁醇溶解。当用2.5%丁醇处理时，来自SV 40转化小鼠细胞的纯化质膜产生T-ag; T-ag从纯化膜中溶解在丁醇中具有温度依赖性，在37°C下提取的T-ag比在22°C下提取的T-ag多约10倍;在细胞表面蛋白用125 I放射性标记后，将2.5%丁醇应用于mKSA细胞导致碘化T-ag的释放。丁醇提取的pmT-ag与p53和几种大小为35至60 kDa的细胞蛋白共沉淀。一种细胞组分以与微管蛋白（56 kDa）相似的迁移率迁移，并且针对微管蛋白a亚基的单克隆抗体共沉淀T-ag。用抗T-ag或p53单克隆抗体免疫沉淀的蛋白质与抗微管蛋白β亚基单克隆抗体的丁醇提取物的免疫印迹显示微管蛋白与T-ag和p53特异性共沉淀。这表明由微管蛋白、T-ag和p53组成的复合物存在于丁醇提取物中。对照实验消除了微管蛋白与丁醇诱导的T-ag和p53的人为关联的可能性。二维凝胶分析显示，2.5%丁醇在37°C下提取了一部分膜相关蛋白和一些胞质蛋白，以及一些不溶于高盐或去污剂的蛋白。因此，本研究中采用的丁醇提取条件回收了似乎与微管蛋白复合的pmT-ag物质。据报道，丁醇对天然蛋白质结构的危害小于其他试剂，包括高盐和去污剂，这种提取方法可能有助于研究其他膜相关蛋白质的结构和功能。
The nature of the interaction of the simian virus 40 (SV40) transforming protein, large tumor antigen (T‐ag), with the plasma membrane of transformed cells is not well understood. We report here that SV40 plasma‐membrane‐associated large tumor antigen (pmT‐ag) can be solubilized by using single‐phase concentrations of 1‐butanol. Purified plasma membranes from SV40‐transformed mouse cells yielded T‐ag when treated with 2.5% butanol; solubilization of T‐ag from the purified membranes in butanol was temperature dependent, with approximately 10‐fold more T‐ag extracted at 37°C than at 22°C; and application of 2.5% butanol to mKSA cells after cellular surface proteins had been radiolabeled with125I resulted in the release of iodinated T‐ag. Butanol‐extracted pmT‐ag coprecipitated with p53 and several cellular proteins ranging in size from 35 to 60 kDa. One cellular component migrated at a mobility similar to that of tubulin (56 kDa), and a monoclonal antibody against the a subunit of tubulin coprecipitated T‐ag. Immunoblotting of proteins immunoprecipitated with monoclonal antibodies against T‐ag or p53 from butanol extracts with a monoclonal antibody against the β subunit of tubulin revealed specific coprecipitation of tubulin with T‐ag and p53. This suggests that complexes composed of tubulin, T‐ag, and p53 exist in butanol extracts. Control experiments eliminated the possibility of an artifactual association of tubulin with T‐ag and p53 induced by butanol. Two‐dimensional gel analyses revealed that 2.5% butanol at 37°C extracted a subset of membrane‐associated proteins and some cytosolic proteins, as well as a number of proteins that were not soluble in either high salt or detergent. Thus, the butanol extraction conditions employed in this study recovered a species of pmT‐ag that appears to complex with tubulin. As butanol reportedly is less deleterious to native protein structures than other agents, including high salts and detergents, this extraction procedure may be useful for studying the structure and function of other membrane‐associated proteins.