46 KDA PROTEIN FROM TREPONEMA DENTICOLA

描述：齿密螺旋体是一种被认为是 在牙周病的发病机制中起重要作用。这项研究旨在 检验一种假说，即来自齿状毛滴虫的46 kDa蛋白作为一种 酶在硫化氢和/或S反应中生成，导致血液氧化 和红细胞破坏的溶血事件。这将通过以下方式完成 1)测定纯化蛋白对红细胞的作用 2)研究了SH基团在46 kDa蛋白介导中的作用 破坏红细胞；3)检测酶的最终产物 以半胱氨酸和谷胱甘肽为底物的反应；4) 确定蛋白质的酶活性是否为吡哆醛 依赖于磷；以及5)研究其他氨基酸是否可以作为 酶的底物。 该蛋白已纯化、克隆、测序并在E. Coli.本申请涉及通过以下方法提纯酶 硫酸铵沉淀、DEAE层析及制备 十二烷基硫酸钠-聚丙烯酰胺凝胶电泳。这种酶将与 绵羊红细胞及其蛋白质随后将通过 十二烷基硫酸钠-聚丙烯酰胺凝胶电泳。为了找出这种酶的产物， 即硫化物，将在毒性作用中发挥重要作用，这种酶 反应将在透析袋中进行，红细胞溶解 在袋子外面发生的情况将用分光光度计量法测量。 蛋白质可能会结合到血细胞膜上 使用免疫金电子显微镜使用抗46 kDa蛋白抗体 是通过用这种蛋白免疫兔子而产生的。 最后，酶的性质将通过表征 反应产物和某些氨基酸作为酶的可能作用 底物使用各种生化技术。

DESCRIPTION: Treponema denticola is one of the spirochetes believed to be important in the pathogenesis of periodontal diseases. The study aims at testing a hypothesis that a 46kDa protein from T. denticola functions as an enzyme in H2S and/or S-generation reactions, resulting in the hemoxidative and hemolytic events of red blood cell destruction. This would be done by 1) determining the action of the purified protein on red blood cell integrity; 2) studying the role of SH-groups in the 46kDa protein-mediated red blood cell destruction; 3) detecting the end-products of the enzyme reaction using cysteine and glutathione as substrates for the protein; 4) finding out if the enzyme activity of the protein is pyridoxal phosphate-dependent; and 5) studying if other amino acids can act as substrate for the enzyme. The protein has been already purified, cloned, sequenced and expressed in E. coli. The present application involves purification of the enzyme by ammonium sulfate precipitation, DEAE-chromatography and preparative SDS-polyacrylamide gel electrophoresis. The enzyme would be incubated with sheep red blood cells and their proteins would be subsequently analyzed by SDS-polyacrylamide gel electrophoresis. To find out if the enzyme products, i.e., sulfur compounds, would be important in the toxic effects, the enzyme reaction would be done inside a dialysis bag and red blood cell lysis occurring outside of the bags would be measured spectrophoto-metrically. Possible binding of the protein to the blood cell membrane would be done using immunogold electron microscopy using anti-46kDa protein antibody that would be produced by immunizing rabbits with the protein. Finally, the nature of the enzyme would be studied by characterizing the reaction products and the possible role of some amino acids as the enzyme substrate using various biochemical techniques.