Studies on the formation of organelles (peroxisomes) in yeast

热带假丝酵母pK 233是一种正构可利用酵母，是研究与细胞器发育有关的特定过氧化物酶体酶定位的优良微生物，因为过氧化物酶体的发育和降解以及过氧化物酶的诱导和抑制可以通过改变生长的碳源来控制。在酵母对正构烷烃的同化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培养的细胞中分离出预先存在的过氧化物酶体，从丙酸培养的细胞中分离出未成熟的过氧化物酶体，从烷烃培养的细胞中分离出成熟的过氧化物酶体，并检测了它们的生化和细胞学特性。酵母细胞中存在的过氧化物酶体是具有乙醛酸循环关键酶的类乙醛酶细胞器。从正构烷烃生长细胞的整个细胞中纯化出仅具有烯酰辅酶A水合酶活性的蛋白质。另一方面，在蛋白酶抑制剂的存在下纯化时，从相同的酵母细胞中获得了具有烯酰辅酶a水合酶和3-羟基酰基辅酶a脱氢酶活性的双功能酶。双功能酶与< α >-糜凝胰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，乙醇-辅酶a水合酶结构域与双功能酶是可分离的，并且在脂肪酸< β - >-氧化系统的演化中是显著的。以过氧化氢酶cDNA为探针，用抗体筛选法分离出一个含有过氧化氢酶基因组DNA序列的克隆。对核苷酸序列和推断的氨基酸序列的分析显示，存在一个明显的区域，负责过氧化氢酶特定定位到过氧化物酶体。

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.