Studies on the formation of organelles (peroxisomes) in yeast

热带假丝酵母PK 233是一种可利用正构烷烃的酵母，是研究与细胞器发育有关的特定过氧化物体酶定位的优良微生物，因为通过改变生长碳源可以很容易地控制过氧化物体的发育和降解以及过氧化物体酶的诱导和抑制。在酵母菌对正构烷烃的同化过程中，观察到成熟的过氧化体的形成。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体，从丙酸生长的细胞中分离出未成熟的过氧化物体，以及从烷烃生长的细胞中分离出成熟的过氧化物体，并对它们的生化和细胞学性质进行了检测。酵母细胞中预先存在的过氧化物体是具有乙醛酸循环关键酶的乙氧体样细胞器。从正构烷烃培养细胞的全细胞中纯化出一种仅具有Enoyl-CoA水合酶活性的蛋白质。另一方面，从相同的酵母细胞中获得了一种具有Enoyl-CoA水合酶和3-羟酰基-CoA脱氢酶活性的双功能酶，该酶在蛋白酶抑制剂的存在下进行纯化。用-lt；α&gt；-糜蛋白酶对该双功能酶进行有限的蛋白降解表明，前者是该双功能酶的一部分。这一事实有力地表明，烯醇化辅酶A的结构域是可以从双功能酶中分离出来的，并且在脂肪酸和β-氧化系统的进化中是值得注意的。用抗体筛选法获得的过氧化氢酶基因作为探针，获得了一个含有过氧化氢酶基因组DNA序列的克隆。对核苷酸序列和推导出的氨基酸序列的分析表明，过氧化氢酶存在一个显著的区域，负责过氧化氢酶在过氧化物体中的特异性定位。

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.