Purification of phosphoenolpyruvate carboxykinase from Saccharomyces cerevisiae and its use for bicarbonate assay

✍ Scribed by Paolo Tortora; Giorgio M. Hanozet; Andrea Guerritore

• Publisher: Elsevier Science
• Year: 1985
• Tongue: English
• Weight: 733 KB
• Volume: 144
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1016/0003-2697(85)90101-0

✦ Synopsis

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 alhnity chromatography on adenosine 5'-monophosphate-Sepharose 4B. In the latter step the binding of the enzyme to the resin specifically required the presence of Mn'+. 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. 0 1985 Academic PUSS, IIIC.