Abstract
This chapter discusses the purification and characterization of galactokinase from Saccharomyces cerevisiae and presents some properties of this enzyme. The enzyme is purified, and characterized from several organisms including Escherichia coli, pig liver, human red cells, and yeast. The purification procedures are carried out at 0–5 ° C and include following steps: preparation of yeast, preparation of extract, ammonium sulfate fractionation, streptomycin sulfate precipitation, diethylaminoethyl (DEAE)-Cellulose chromatography, hydroxyapatite chromatography, and BioGel A-0.5m gel filtration. Several properties of the enzyme, such as purity, stability, structure, catalysis, and immunology are discussed in the chapter. The results show that this enzyme gives anomalous results when run on polyacrylamide gels in the absence of sodium dodecyl sulfate (SDS). The enzyme has a pH optimum at 8.0 with a sharp drop in activity above pH 9.0 and a more gradual drop below pH 7.0. The antisera prepared against the purified enzyme does not cross-react with E. coli galactokinse and the antisera against the E. coli enzyme does not cross-react with the yeast enzyme.
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