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

古细菌热原体细胞骨架和脂质生物合成系统的研究

• 批准号: 09839033
• 负责人: YAMAGISHI Akihiko
• 金额: $ 1.73万
• 依托单位: TOKYO UNIVERSITY OF PHARMACY AND LIFE SCIENCE
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1997
• 资助国家: 日本
• 起止时间: 1997 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09839033/
• 关键词: Thermoplasma; cytoskeleton; Archaea; archaebacteria; etherlipid; terbinafine; tetraetherlipid; Thermoplasma; 原子間力顕微鏡; テルビナフィン; テトラエーテル; ジエーテル; 好熱好酸古細菌

(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.

(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。

项目成果

deBruijn,F.,J.: "Bcterial Genomes:Physical Structure and Analysis" Chapman&Hall,New York, 793 (1997)

deBruijn,F.,J.：“细菌基因组：物理结构和分析”查普曼

古賀洋介 編: "古細菌の生物学" 東京大学出版会, 302 (1998)

古贺洋介编：《古菌生物学》东京大学出版社，302（1998）

Motono, C.,: "Urea-induced unfolding and conformational stability of 3-isopropylmalate dehydrogenase from the thermophile Thermus thermophilus and its mesophilic counterpart from Escherichia coli."Biochemistry. 38. 1332-1337 (1999)

Motono, C.，：“来自嗜热栖热菌和来自大肠杆菌的嗜温对应物的 3-异丙基苹果酸脱氢酶的尿素诱导的展开和构象稳定性。”生物化学。

山岸明彦: "進化分子工学を利用した蛋白質分子育種の実例 3)耐熱性酵素の低温適応"化学と生物. 38. 118-121 (2000)

Akihiko Yamagishi：“利用进化分子工程进行蛋白质分子育种的示例 3) 耐热酶的低温适应”化学与生物学，38. 118-121 (2000)。

Tamakoshi, M., Yaoi T., Oshima, T., and Yamagishi, A.: "An efficient gene replacement and deletion system for an extreme thermophile, Thermus thermophilus."FEMS Microbiol. Lett.. 173. 431-437 (1999)

Tamakoshi, M.、Yaoi T.、Oshima, T. 和 Yamagishi, A.：“一种针对极端嗜热菌、嗜热栖热菌的高效基因替换和删除系统。”FEMS Microbiol。