Christeller J T, Tolbert N E
J Biol Chem. 1978 Mar 25;253(6):1780-5.
Phosphoglycolate phosphatase (EC 3.1.3.18) was purified 1500-fold from field-grown tobacco leaves by acetone fractionation, DEAE-cellulose and molecular sieve chromatography, and preparative polyacrylamide gel electrophoresis. Preparations were judged 90 to 95% homogeneous by chromatography on DEAE-cellulose, polyacrylamide gel electrophoresis, and by isoelectric focusing. The highest specific activity obtained was 468 mumol of phosphate released/min/mg of protein. The native protein has a molecular weight of 80,500 by Ferguson plot analysis and 86,300 by sedimentation velocity on sucrose density gradients. Sodium dodecyl sulfate-polyacrylamide gels gave a molecular weight of 20,700, indicating the P-glycolate phosphatase is a tetramer with identical or near identical subunits. The enzyme, freshly purified or in crude homogenates, had a pI of 3.8 to 3.9 pH units by isoelectric focusing. Phosphosphoglycolate phosphatase from spinach leaves has a molecular weight of 93,000 and, unlike the enzyme from tobacco leaves, it is extremely unstable after DEAE-cellulose chromatography and is inactivated by lipase (EC 3.1.1.3). The phosphatase from both plants was stabilized by the addition of citrate or isocitrate in the buffers. Ribose 5-phosphate is a competitive inhibitor of phosphoglycolate phosphatase at physiological concentration, while other phosphate esters of the photosynthetic carbon cycle were without effect.
通过丙酮分级分离、DEAE-纤维素和分子筛色谱法以及制备型聚丙烯酰胺凝胶电泳，从田间种植的烟草叶片中纯化出磷酸乙醇酸磷酸酶（EC 3.1.3.18），纯化倍数达1500倍。通过DEAE-纤维素柱色谱、聚丙烯酰胺凝胶电泳和等电聚焦分析，判断制备物的纯度为90%至95%。获得的最高比活性为每分钟每毫克蛋白质释放468微摩尔磷酸盐。通过Ferguson作图分析，天然蛋白质的分子量为80,500，通过蔗糖密度梯度沉降速度法测得为86,300。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳测得分子量为20,700，表明磷酸乙醇酸磷酸酶是由相同或近乎相同的亚基组成的四聚体。通过等电聚焦分析，新鲜纯化的或粗匀浆中的该酶的等电点为3.8至3.9个pH单位。菠菜叶中的磷酸乙醇酸磷酸酶分子量为93,000，与烟草叶中的酶不同，它在DEAE-纤维素色谱后极不稳定，且会被脂肪酶（EC 3.1.1.3）灭活。两种植物来源的磷酸酶在缓冲液中添加柠檬酸盐或异柠檬酸盐后均得到稳定。在生理浓度下，5-磷酸核糖是磷酸乙醇酸磷酸酶的竞争性抑制剂，而光合碳循环中的其他磷酸酯则无此作用。
