Purification and Characterization of a Galactose-1-Phosphate: UDP-Glucose Uridyltransferase from the Red Alga Galdieria Sulphuraria

Authors

  • Wolfgang Gross,

    Corresponding author
    1. Freie Universität Berlin, Institut für Pflanzenphysiologie und Mikrobiologie, Berlin, Germany
      W. Gross, Freie Universität Berlin, Institut für Pflanzenphysiologie und Mikrobiologie, Königin-Luise-Str. 12–16a, D-14195 Berlin, Germany
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  • Claus Schnarrenberger

    1. Freie Universität Berlin, Institut für Pflanzenphysiologie und Mikrobiologie, Berlin, Germany
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W. Gross, Freie Universität Berlin, Institut für Pflanzenphysiologie und Mikrobiologie, Königin-Luise-Str. 12–16a, D-14195 Berlin, Germany

Abstract

The galactose-1-phosphate uridyltransferase of the red alga Galdieria sulphuraria has been purified about 1800-fold to a final specific activity of approximately 140 U/mg protein. The purification involved chromatography on DEAE-Fractogel, hydroxyapatite, decyl-agarose, and DEAE-Tentacle gel. After SDS/PAGE, the enzyme preparation showed only one protein band of 42 kDa. The enzyme is a homodimer with a molecular mass of 82 kDa as estimated from the sedimentation velocity or 60 kDa as estimated by gel filtration. It has a broad pH optimum between pH 7 and pH 9. The apparent Km values for the forward and backward reactions are Km(Glc1P) = 105 μM, Km(UDP-galactose) = 30 μM, Km(Gal1P) = 400 μM, and Km(UDP-Glc) = 20 μM. The activation energy of the reaction is 45 kJ mol-1. The enzyme is specific for the galactose 1-phosphate to UDP-galactose interconversion in the Leloir pathway while the alternate enzyme for the Isselbacher pathway, UDP-galactose pyrophosphorylase, could not be detected in G. sulphuraria.

Enzymes
 

Galactose-1-phosphate uridyltransferase (EC 2.7.7.12)

 

UDP-galactose pyrophosphorylase (EC 2.7.7.10)

 

UDP-glucose pyrophosphorylase (EC 2.7.7.9)

Ancillary

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