46 KDA PROTEIN FROM TREPONEMA DENTICOLA

描述：齿垢密螺旋体是被认为是 在牙周病的发病机制中起重要作用。 该研究旨在 验证了一个假设，即来自T.齿垢的功能是 H2S和/或S-生成反应中的酶，导致血氧化 和红细胞破坏的溶血性事件。 这将由以下人员完成： 1)测定纯化蛋白对红细胞的作用 2）研究SH-基团在46 kDa蛋白介导的 红细胞破坏; 3）检测酶的终产物 使用半胱氨酸和谷胱甘肽作为蛋白质的底物的反应; 4） 发现蛋白质的酶活性是否是吡哆醛 磷酸盐依赖性; 5）研究其他氨基酸是否可以作为 酶的底物。 该蛋白已被纯化、克隆、测序并在大肠杆菌中表达。 杆菌 本申请涉及通过以下方法纯化酶 硫酸铵沉淀、DEAE-色谱和制备 SDS-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 SDS-聚丙烯酰胺凝胶电泳。 为了找出酶的产物， 也就是说，硫化合物，将是重要的毒性作用，酶 反应将在透析袋内进行， 发生在袋外的情况将通过分光光度法测量。 蛋白质与血细胞膜的可能结合将在 使用免疫金电子显微镜，使用抗46 kDa蛋白抗体， 将通过用这种蛋白质免疫兔子来产生。 最后，将通过表征酶的性质来研究酶的性质。 反应产物和一些氨基酸作为酶的可能作用 使用各种生物化学技术的底物。

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.