Study of the cytoskeleton and lipid biosynthetic system of an archaeon Thermoplasma

(1)对热浆菌的类细胞色素结构进行了生化分析。用非离子去污剂Triton X-100处理热浆菌细胞。通过阴离子交换柱层析和凝胶排阻层析的组合，对溶解的蛋白质进行分级。回收了几种可以可逆降解的蛋白质组分。电镜观察发现部分组分中存在管状结构和网状结构。通过SDS-聚丙烯酰胺凝胶电泳分析组分的肽组成。(2)用14碳标记的甲羟戊酸追踪热浆菌醚膜脂的生物合成途径。从C14-甲羟戊酸存在下生长的细胞中提取热浆菌的极性醚-脂质部分。提取的醚脂通过二维薄层色谱法进行分析。从TLC板的各个点回收脂质，并分析和估计脂质的结构。孵育2小时后，主要的极性四醚脂质，其中有磷脂酰甘油和糖分子，积累在细胞中。在抑制剂特比萘芬的存在下，主要极性脂质的生物合成被抑制，并且二醚脂质被积累。去除抑制剂后，通过薄层色谱法分析极性脂质的生物合成。提出了四脂生物合成途径的模型。

(1) Cytoskeleton-like structures of Thermoplasma were biochemically analyzed. Cells of Thermoplasma were treated by non-ionic detergent Triton X-100. The solubilized proteins were fractionated by the combination of anion-exchange-column chromatography and gel-exclusion chromatography. Several fractions of proteins that can reversibly polymerize were recovered. Tubular structures and net-like structures were found in some of those fractions by electron microscopic inspection. Peptide composition of the fractions was analyzed by SDS-polyacrylamide gel electophoresis.(2) Carbon 14-labeled mevalonic acid was used to trace the biosynthetic pathway of ether-membrane lipids in Thermoplasma. Polar ether-lipid fraction of Thermoplasma was extracted from the cells grown in the presence of C14-mevalonic acid. The extracted ether-lipids were analyzed by two-dimensional thin layer chromatography. Lipids were recovered from respective spot of TLC plates and the structures of the lipids were analyzed and estimated. After 2-h incubation, major polar tetraetherlipid, which has phosphatidylglycerol and a sugar molecule, was accumulated in the cells. In the presence of the inhibitor terbinafine, biosynthesis of the major polar lipid was inhibited and a diether lipid was accumulated. After the removal of the inhibitor, biosynthesis of polar lipids was analyzed by thin layer chromatography. The model of the biosynthetic pathway of the tetra-lipids was proposed.