Abstract
This chapter provides an overview of assay method, purification procedure, and the properties of phosphoglycerate kinase from Bacillus stearothermophilus . 1,3-Diphosphoglycerate formed from ATP and 3-phosphoglycerate is converted to glyceraldehydes-3-phosphate by a coupled enzyme assay with glyceraldehyde-3-phosphate dehydrogenase in the presence of NADH. The concomitant oxidation of NADH is measured spectrophotometrically as the decrease of absorption at 340 nm. The purification procedure involves extraction, streptomycin treatment, ammonium sulfate fraction, diethylaminoethyl (DEAE)-cellulose chromatography, gel filtration, and further purification by rechromatography on Sephadex G-100 or hydroxyapatite column chromatography. The enzyme purified by the above procedure is homogeneous as judged by polyacrylamide gel electrophoresis with or without sodium dodecyl sulfate (SDS) and by electrophoresis on cellulose acetate strips at pH 3.9–8.6. The molecular weight of the enzyme is 42,000, based on the results of gel filtration, SDS-polyacrylamide gel electrophoresis, and amino acid analyses. The enzyme shows a broad pH activity profile with maximum activity at pH 5.5–8.5.
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