Elucidation of acid tolerant function of Thiobacillus thiooxidans grown under scid and extreme environment

阐明在SCID和极端环境下生长的氧化硫硫杆菌的耐酸功能

• 批准号: 09650871
• 负责人: AMANO Yoshifumi
• 金额: $ 2.18万
• 依托单位: Yamanashi University
• 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 1998
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09650871/
• 关键词: Thiobacillus thiooxidans; Axid Tolerance; Cytochrome c oxidase; Membrane-bound cytochrome c; シトクロムcオキシダーゼ; シトクロム c オキシダーゼ

We studied isolations and properties of cytochrome c oxidase and membrane-bound cytochrome C to elucidate acid tolerant function of Thiobactillus thiooxidans grown under acid and extreme environment.1. Purification of cytochrome c oxidase : Cell suspension of T.thiooxidans was disrupted and the homogenate was centrifuged to prepare the cell-free extracts. The membrane fraction was prepared by centrifuging the cell-free extracts. The activity of cytochrome C oxidase was estimated by measuring the decrease rate of the absorbance at 550nm due to the oxidation of reduced cytochrome c. Enzymatic properties of the membrane fractions were studied. Optimum pH and temperature of the enzyme reaction was 5.0 and 350C, respectively. The enzyme activity of the membrane fractions was stable at pH 4.0. The enzyme was solubilized with Triton X-100 from the membrane fractions. The Isoelectric point of the enzyme in the solubilized fraction was found to be 5.5 by detection of the indophenol blue band on … More the isoelectric focusing gel. The solubulized fraction was subjected to CM-cellulose and Sephacryl S-200 colums In the presence of Triton X- 100 at pH 4.0. The purified enzyme fraction had two protein bands by native PAGE.Therefore, the enzyme preparation was not homogeneous.2. Purification of membrane-bound cytochrome C : The fraction solubilized with Triton X- 100 from membrane fraction was subjected to isoelectric focusing. By staining the heme protein bands on the gel, it was found that the solubilized fraction was composed of three different cytochrome c proteins, with isoelectric points of 5.3, 5.8 and 8.2, respectively. The acidic proteins (p1 5.3 and p1 5.8) were subjected to DEAE-Sepharose column in the presence of Triton X- 100 at pH 7.0. However, the protein exihibiting an absorption spectrum of cytochrome c was not observed in the eluted fractions. The basic protein (p1 8.2) was chromatographed with CM-cellulose and hydroxyapatite colums in the presence of Triton X- 100 at pH 7.0. A protein exihibiting the absorption spectrum of cytochrome c was observed in the eluted fractions. The cytochrome c fraction was homogeneous on SDS-PAGE, The molecular weight of the membrane-bound cytochrome c was estimated to be 27kD. Less

本研究通过对细胞色素C氧化酶和膜结合细胞色素C的分离和性质的研究，阐明氧化硫硫杆菌在酸性和极端环境下的耐酸功能.细胞色素c氧化酶的纯化：将氧化硫杆菌的细胞悬浮液破碎，并将匀浆离心以制备无细胞提取物。通过离心无细胞提取物制备膜级分。通过测量由于还原的细胞色素c的氧化而导致的在550 nm处的吸光度的降低速率来估计细胞色素C氧化酶的活性。对膜组分的酶性质进行了研究。酶反应的最适pH为5.0，最适温度为350 ℃。膜组分的酶活性在pH 4.0下是稳定的。用Triton X-100从膜级分中溶解酶。通过检测上的靛酚蓝条带，发现溶解级分中的酶的等电点为5.5。 ...更多信息 等电聚焦凝胶。在pH 4.0的Triton X- 100存在下，将溶解的部分通过CM-纤维素和Sephacryl S-200柱。纯化后的酶组分经非变性聚丙烯酰胺凝胶电泳显示有两条蛋白带，因此酶制剂不具有特异性.膜结合细胞色素C的纯化：用Triton X- 100从膜级分中溶解的级分进行等电聚焦。通过对凝胶上的血红素蛋白条带进行染色，发现溶解级分由三种不同的细胞色素c蛋白组成，其等电点分别为5.3、5.8和8.2。在pH 7.0的Triton X- 100存在下，将酸性蛋白（p1 5.3和p1 5.8）上DEAE-Sepharose柱。然而，在洗脱组分中未观察到显示细胞色素c吸收光谱的蛋白质。碱性蛋白质（p1 8.2）在pH 7.0的Triton X- 100存在下，用CM-纤维素和羟基磷灰石柱进行色谱分离。在洗脱组分中观察到一种具有细胞色素c吸收光谱的蛋白质。SDS-聚丙烯酰胺凝胶电泳显示细胞色素c组分为均一组分，其分子量约为27 kD。少