Abstract
This chapter provides an overview of assay method, purification procedure, and the properties of 3-phosphoglycerate kinase from bovine liver and yeast. 3-Phosphoglycerate kinase activity is assayed spectrophotometrically at 366 nm in a test system coupled with D-glyceraldehyde-3-phosphate dehydrogenase. Purification of bovine liver 3-phosphoglycerate kinase involves preparation of crude extract and ammonium sulfate fractionation, heat denaturation in the presence of magnesium sulfate, diethylaminoethyl (DEAE)-cellulose batch chromatography, Sephadex G-75 gel filtration, DEAE-Sephadex A-50 ion-exchange chromatography, and Sephadex G-l00 gel filtration. In the purification of yeast 3-phosphoglycerate kinase, anion-exchange and hydroxyapatite chromatography are replaced by reversible salting-out chromatography on Sepharose CL-6B. Dye-substituted Sepharose CL-6B and Sephadex G-100 are not well suited for purification of yeast phosphoglycerate kinase.
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