Purification and properties of galactokinase from Tetrahymena thermophila

Joel E. Lavine, Eric Cantlay, Charles T. Roberts, Daniel E. Morse

    Research output: Contribution to journalArticle

    15 Scopus citations

    Abstract

    Galactokinase (EC 2.7.1.6; ATP: d-galactose-1-phosphototransferase) was purified 152-fold with an 11% yield from Tetrahymena thermophila maximally derepressed for enzyme synthesis in late stationary phase. The purification procedure utilized sequential acid precipitation, batch DEAE-Sephacel chromatography, differential ammonium sulfate precipitation and narrow range electrofocusing. The apparent molecular weight of the holoenzyme as determined by gel filtration on Sephadex G-200 is 50 000-55 000. The holoenzyme consists of two subunits of approx. 28 000 daltons each, as determined by SDS-polyacrylamide gel electrophoresis. The native enzyme appears to be a single species with an isoelectric point at pH 5.1 Optimal activity was obtained at pH 7.8 and 41°C, with no added monovalent salt. d-Galactose, 2-deoxygalactose and galactosamine all are suitable carbohydrate substrates for the stereospecific galactokinase; only substitution at the C-2 position of galactose retains enzyme recognition. The enzyme utilizes ATP, 2′-dATP and 3′-dATP as phosphate donors; ADP and adenosine-5′-[γ-thio]triphosphate are inhibitory. The Km values for galactose and ATP were determined to be 0.60 mM and 0.15 mM, respectively. The enzyme requires a divalent cation for activity, with effectiveness being in the order: Mg2+ >Co2+ >Mn2+ >Fe2+. Galactokinases from all eucaryotic sources studied thus far seem to be very similar. Based upon the results reported here, the galactokinases from Tetrahymena and yeast appear to be most similar in their biophysical and biochemical properties.

    Original languageEnglish (US)
    Pages (from-to)76-85
    Number of pages10
    JournalBBA - General Subjects
    Volume717
    Issue number1
    DOIs
    StatePublished - Jul 16 1982

      Fingerprint

    Keywords

    • (T. thermophila)
    • Galactokinase
    • Substrate specificity

    ASJC Scopus subject areas

    • Biophysics
    • Biochemistry
    • Molecular Biology

    Cite this