Abstract
Acetate kinase is an enzyme found only in microorganisms and functions in the metabolism of pyruvate or synthesis of acetyl-CoA coupling with phosphoacetyltransacetylase. This chapter describes an assay method for the formation of adenosine diphosphate (ADP) from acetate and adenosine triphosphate (ATP) that is measured spectrophotometrically after NADH oxidation in the presence of pyruvate kinase (PK) and lactate dehydrogenase (LDH). The chapter also discusses the purification procedure of acetate kinase from Bacillus stearothermophilus and properties of this enzyme. The purification process involves growth of bacterial cells and preparation of hydroxyapatite that includes extraction of enzyme, streptomycin treatment, ammonium sulfate fractionation, diethylaminoethyl (DEAE)-cellulose column chromatography, hydroxyapatite column chromatography, ultrogel chromatography, and DEAE-Sephadex A-50 chromatography followed by crystallization. The purified enzyme is homogeneous as judged by polyacrylamide gel electrophoresis with or without sodium dodecyl sulfate (SDS) and by cellulose acetate strip electrophoresis at pH 5.5–8.5. The molecular weight estimated by gel filtration and sedimentation analyses is 160,000–170,000.
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