Purification and Characterization
Purification and Characterization
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DOI：
10.1002/0470080124.ch3
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发表时间：
2006-04
影响因子：
5.6
通讯作者：
M. Torruella;M. Flawiá;C. Eisenschlos;L. M. Vedia;C. Rubinstein;H. Torres
中科院分区：
文献类型：
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作者：
M. Torruella;M. Flawiá;C. Eisenschlos;L. M. Vedia;C. Rubinstein;H. Torres
腺苷酸环化酶活性与克氏锥虫可沉淀的馏分溶解处理与非离子型洗涤剂Lubrol PX和0.5 M-（NH 4）2SO 4。以下流体动力学和分子参数建立了部分纯化的酶-洗涤剂复合物：沉降系数6.2 S;斯托克斯半径5.65 nm;部分比容0.83 ml/g; Mr 244000;摩擦比1.33。从这些参数中计算出无洗涤剂蛋白质的Mr约为124000。该酶活性的pI为6.2。制备了抗T.获得了Cruzi腺苷酸环化酶，其抑制来自几种低等真核生物的环化酶活性。霸王通过在免疫亲和层析柱中使用该抗体进一步纯化Cruzi腺苷酸环化酶。在SDS/聚丙烯酰胺凝胶电泳上，该层析后获得的级分显示具有约56000的表观Mr的主要多肽带，其与单克隆抗体特异性反应。
Adenylate cyclase activity associated with Trypanosoma cruzi sedimentable fractions was solubilized by treatment with the non-ionic detergent Lubrol PX and 0.5 M-(NH4)2SO4. The following hydrodynamic and molecular parameters were established for a partially purified enzyme-detergent complex: sedimentation coefficient 6.2 S; Stokes radius 5.65 nm; partial specific volume 0.83 ml/g; Mr 244000; frictional ratio 1.33. A Mr of about 124000 was calculated for the detergent-free protein from these parameters. The pl of this enzyme activity was 6.2. A monoclonal antibody to T. cruzi adenylate cyclase was obtained, which inhibited cyclase activities from several lower eukaryotic organisms. The T. cruzi adenylate cyclase was further purified by using this antibody in immunoaffinity chromatographic columns. Fractions obtained after this chromatography showed, on SDS/polyacrylamide-gel electrophoresis, a main polypeptide band with an apparent Mr of about 56000, which specifically reacted with the monoclonal antibody.