Strobel R S, Nagy A K, Knowles A F, Buegel J, Rosenberg M D
Department of Genetics and Cell Biology, University of Minnesota, St. Paul, Minnesota 55108, USA.
J Biol Chem. 1996 Jul 5;271(27):16323-31. doi: 10.1074/jbc.271.27.16323.
An ecto-ATP diphosphohydrolase (ATPDase) was purified to homogeneity from vesiculosomes shed from chicken oviduct. First, the ecto-ATPDase-enriched vesiculosomes were concentrated by filtration, differential centrifugation, and exclusion chromatography. Next, the nonionic detergent, Nonidet P-40, was used to extract the ecto-ATPDase from vesiculosomal membranes, and the solubilized enzyme was further purified by ion exchange (DEAE-Bio-Gel) and lentil-lectin-Sepharose 4B chromatography. In the final stage, immunoaffinity chromatography was utilized to obtain purified ecto-ATPDase. More than 25,000-fold purification was achieved. Specific activity of the purified enzyme was greater than 800 micronol/min/mg of protein with MgATP as the substrate, the highest ever reported for an ATPDase. The enzyme also hydrolyzed other nucleoside triphosphates in the presence of magnesium at similar rates and CaATP and MgADP at lower rates. The molecular mass of the purified glycoprotein was 80 kDa as determined by SDS-polyacrylamide gel electrophoresis and Western blot analysis. Based on its enzymatic properties, the relationship of the chicken oviduct ecto-ATPDase with other reported ATPDases and ecto-ATPases is discussed.
从鸡输卵管脱落的囊泡小体中纯化出一种胞外ATP二磷酸水解酶（ATPDase），使其达到同质状态。首先，通过过滤、差速离心和排阻色谱法浓缩富含胞外ATPDase的囊泡小体。接下来，使用非离子去污剂Nonidet P - 40从囊泡小体膜中提取胞外ATPDase，然后通过离子交换（DEAE - Bio - Gel）和扁豆凝集素 - 琼脂糖4B色谱法进一步纯化溶解的酶。在最后阶段，利用免疫亲和色谱法获得纯化的胞外ATPDase。实现了超过25000倍的纯化。以MgATP为底物时，纯化酶的比活性大于800微摩尔/分钟/毫克蛋白质，这是ATPDase报道的最高比活性。该酶在镁存在下也能以相似速率水解其他核苷三磷酸，对CaATP和MgADP的水解速率较低。通过SDS - 聚丙烯酰胺凝胶电泳和蛋白质印迹分析测定，纯化糖蛋白的分子量为80 kDa。基于其酶学性质，讨论了鸡输卵管胞外ATPDase与其他已报道的ATPDase和胞外ATP酶的关系。
