杜氏利什曼原虫3'-核苷酸酶在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳后的复性

Renaturation of Leishmania donovani 3'-nucleotidase following sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Zlotnick G W, Mackow M C, Gottlieb M

Comp Biochem Physiol B. 1987;87(3):629-35. doi: 10.1016/0305-0491(87)90063-0.

Abstract

Renaturation of a 3'-nucleotidase from the surface membrane of Leishmania donovani promastigotes was achieved following polyacrylamide gel electrophoresis (PAGE) in the presence of sodium dodecyl sulfate (SDS). 2. Enzyme activity was detected in situ in gels, following SDS removal, by incubating the gels in reaction mixtures containing 3'-AMP or 3'-UMP as substrate followed by staining for the inorganic phosphate (Pi) reaction product with malachite green-molybic acid solution. 3. Conditions for the removal of SDS by diffusion and for the renaturation of enzyme activity are described including evidence for the detergent requirement, which is best satisfied by 3[(3-cholamidopropyl)-dimethylammonio]2-hydroxy-1-propane sulfonate (CHAPSO). 4. Results indicate that the 3'-nucleotidase migrates under these conditions as a polypeptide with an Mr of 43,000.

摘要

在十二烷基硫酸钠（SDS）存在的情况下，通过聚丙烯酰胺凝胶电泳（PAGE）实现了来自杜氏利什曼原虫前鞭毛体表面膜的3'-核苷酸酶的复性。2. SDS去除后，通过将凝胶在含有3'-AMP或3'-UMP作为底物的反应混合物中孵育，然后用孔雀石绿-钼酸溶液对无机磷酸盐（Pi）反应产物进行染色，在凝胶中原位检测酶活性。3. 描述了通过扩散去除SDS和酶活性复性的条件，包括对去污剂需求的证据，3[(3-胆酰胺丙基)-二甲基铵基]2-羟基-1-丙烷磺酸盐（CHAPSO）最能满足该需求。4. 结果表明，在这些条件下，3'-核苷酸酶作为一种Mr为43,000的多肽迁移。