Tortora P, Hanozet G M, Guerritore A
Anal Biochem. 1985 Jan;144(1):179-85. doi: 10.1016/0003-2697(85)90101-0.
Electrophoretically homogeneous phosphoenolpyruvate carboxykinase (EC 4.1.1.49) from Saccharomyces cerevisiae was obtained in high yields by means of a two-step purification procedure consisting of ion-exchange chromatography and affinity chromatography on adenosine 5'-monophosphate-Sepharose 4B. In the latter step the binding of the enzyme to the resin specifically required the presence of Mn2+. The enzyme was eluted when Mn2+ was removed by addition of ethylenediaminetetraacetate to the elution buffer. Homogeneity, molecular weight, and subunit composition of phosphoenolpyruvate carboxykinase were checked by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration. A factor which caused an underestimation of the enzyme activity in crude extracts was identified as adenylate kinase. Finally, a method is proposed for the enzymatic assay of bicarbonate using a purified phosphoenolpyruvate carboxykinase preparation.
通过两步纯化程序，即离子交换色谱法和在5'-单磷酸腺苷-琼脂糖4B上的亲和色谱法，从酿酒酵母中以高产率获得了电泳纯的磷酸烯醇丙酮酸羧激酶（EC 4.1.1.49）。在第二步中，酶与树脂的结合特别需要Mn2+的存在。当在洗脱缓冲液中加入乙二胺四乙酸除去Mn2+时，酶被洗脱。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和凝胶过滤检查了磷酸烯醇丙酮酸羧激酶的均一性、分子量和亚基组成。在粗提物中导致酶活性低估的一个因素被鉴定为腺苷酸激酶。最后，提出了一种使用纯化的磷酸烯醇丙酮酸羧激酶制剂对碳酸氢盐进行酶促测定的方法。
