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Thermoplasma的类细胞骨架结构进行了生化分析。 Thermoplasma 细胞用非离子去污剂 Triton X-100 处理。通过阴离子交换柱色谱法和凝胶排阻色谱法的组合对溶解的蛋白质进行分级。回收了几种可以可逆聚合的蛋白质。通过电子显微镜检查，在其中一些级分中发现了管状结构和网状结构。采用SDS-聚丙烯酰胺凝胶电泳分析各组分的肽组成。(2)采用碳14标记的甲羟戊酸追踪热原体中醚膜脂质的生物合成途径。 Thermoplasma 的极性醚-脂质部分是从在 C14-甲羟戊酸存在下生长的细胞中提取的。通过二维薄层色谱分析提取的醚-脂质。从薄层色谱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，含有磷脂酰甘油和糖分子的主要极性四醚脂在细胞中积累。在抑制剂特比萘芬存在下，主要极性脂质的生物合成受到抑制，并且二醚脂质积累。除去抑制剂后，通过薄层色谱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。