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Studies on the formation of organelles (peroxisomes) in yeast

酵母细胞器（过氧化物酶体）形成的研究

基本信息

批准号：
61550723
负责人：
TANAKA Atsuo
金额：
$ 1.22万
依托单位：
Kyoto University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1986
资助国家：
日本
起止时间：
1986 至 1987
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-61550723/
关键词：
n-Alkane-utilizable yeast Candida tropicalis pK 233 Peroxisomes Fatty acid <beta>-oxidation system Bifunctional enzyme Domain of enoly-CoA hydratese Genomic DNA sequence for the peroxisomal catalase 局在化炭化水素資化性酵母エノイル-CoAヒドラターゼ 3-ヒドロキシ

项目摘要

Candida tropicalis pK 233, an n-alkane-utilizable yeast, is an excellent microorganism for studying the localization of specific peroxisomal enzymes in connection with the development of the organelles since development and degradation of peroxisomes as well as the induction and repression of peroxisomal enzymes can be easily controlled by changing the carbon source for growth.1. Development of the mature peroxisomes was observed during the assimilation of n-alkanes by the yeast. We succeeded in the isolation of the pre-existing peroxisomes from glucose-grown cells and the immature ones from propionate-grown cells as well as the mature ones from alkane-grown cells, and examined their biochemical and cytological properties. The pre-existing peroxisomes form the yeast cells were found to be glyoxysome-like organelles having the key enzymes of glyoxylate cycle.2. A protein exhibiting only enoyl-CoA hydratase activity was purified from the whole cells of n-alkane-grown cells. On the other hand, a bifunctional enzyme exhibiting enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase activities was obtaind from the same yeast cells when purified in the presence of protease inhibitors. Limited proteolysis of the bifunctional enzyme with <alpha>-chymotrypsin indicated that the former was a part of the bifunctional enzyme. The fact strongly suggests that the domain of enoly-CoA hydratese is separable from the bifunctional enzyme and is noticeable on the evolution of fatty acid <beta>-oxidation system.3. A clone harbouring the genomic DNA sequence for the peroxisomal catalase has been isolated using the catalase cDNA as a probe, which had been obtained with the antibody-screening method. Analysis of the nucleotide sequence and the deduced amino acid sequence revealed the presence of a noticeable region responsible to the specific localization of catalase into peroxisomes.

热带假丝酵母 pK 233 是一种可利用正烷烃的酵母，是研究与细胞器发育相关的特定过氧化物酶体酶定位的优秀微生物，因为过氧化物酶体的发育和降解以及过氧化物酶体酶的诱导和抑制可以通过改变生长碳源轻松控制。1。在酵母同化正烷烃的过程中观察到成熟过氧化物酶体的发育。我们成功地从葡萄糖生长的细胞中分离出预先存在的过氧化物酶体，从丙酸盐生长的细胞中分离出未成熟的过氧化物酶体，以及从烷烃生长的细胞中分离出成熟的过氧化物酶体，并检查了它们的生化和细胞学特性。研究发现酵母细胞中预先存在的过氧化物酶体是类乙醛酸酶体的细胞器，具有乙醛酸循环的关键酶。 2．从正烷烃生长的细胞的全细胞中纯化出仅表现出烯酰辅酶A水合酶活性的蛋白质。另一方面，当在蛋白酶抑制剂存在下纯化时，从相同的酵母细胞中获得了表现出烯酰辅酶A水合酶和3-羟酰辅酶A脱氢酶活性的双功能酶。双功能酶与α-胰凝乳蛋白酶的有限蛋白水解表明前者是双功能酶的一部分。这一事实有力地表明烯醇辅酶A水合酶的结构域与双功能酶是可分离的，并且在脂肪酸β-氧化系统的进化中具有显着的作用。 3．使用通过抗体筛选方法获得的过氧化氢酶cDNA作为探针，分离了含有过氧化物酶体过氧化氢酶基因组DNA序列的克隆。对核苷酸序列和推导的氨基酸序列的分析揭示了一个值得注意的区域的存在，该区域负责过氧化氢酶特异性定位到过氧化物酶体中。