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

我们研究了细胞色素c氧化酶和膜结合细胞色素c的分离和性质，以阐明生长在酸性和极端环境下的硫代氧化硫杆菌的耐酸功能。细胞色素c氧化酶的纯化：破坏T.thiooxidans细胞悬液，离心匀浆，制备无细胞提取物。将无细胞提取物离心制备膜组分。通过测定在550nm处细胞色素C氧化引起的吸光度降低率来测定细胞色素C氧化酶的活性，并研究了膜组分的酶学性质。酶反应的最适pH为5.0，最适温度为350C。膜组分的酶活性在pH 4.0时稳定。该酶用Triton X-100从膜组分中溶解。通过等电聚焦凝胶上的吲酚蓝带检测，发现溶出部分酶的等电点为5.5。在Triton X- 100存在下，pH为4.0，将溶解后的馏分用cm -纤维素和sepphacryl S-200色谱柱测定。经天然PAGE分析，纯化后的酶片段有两条蛋白带。因此，酶制剂不均匀。膜结合细胞色素C的纯化：用Triton X- 100溶解的膜组分进行等电聚焦。通过对凝胶上血红素蛋白条带的染色，发现溶解部分由三种不同的细胞色素c蛋白组成，等电点分别为5.3、5.8和8.2。酸性蛋白（p1 5.3和p1 5.8）在Triton X- 100存在下，pH 7.0进行DEAE-Sepharose色谱柱分析。然而，在洗脱的组分中没有观察到蛋白质对细胞色素c的吸收光谱。在Triton X- 100存在下，pH 7.0，用cm -纤维素和羟基磷灰石柱对碱性蛋白（p1 8.2）进行色谱分析。在洗脱的组分中观察到一种具有细胞色素c吸收光谱的蛋白。细胞色素c在SDS-PAGE上均相，膜结合细胞色素c的分子量估计为27kD。少

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