Purification and characterization of UDP-D-galactose 4-epimerase from the red alga Galdieria sulphuraria

UDP-D-galactose 4-epimerase of the unicellular red alga Galdieria sulphuraria has been purified to apparent electrophoretic homogeneity by chromatography on DEAE-Fractogel, hydroxylapatite and by affinity chromatography on Dyematrex Orange. The holoenzyme is a homodimer with an apparent molecular mass of 83 and 76 kDa as determined by gelfiltration and by sucrose gradient centrifugation, respectively. The size of the subunits was 42 kDa as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The 4-epimerase from G. sulphuraria does not require external NAD for activity, unlike the enzyme from some other organisms, and inhibition by NADH was not observed. The apparent K-m, for UDP-D-galactose was 64 mu M. The pH optimum was at 8 and the apparent equilibrium constant for UDP-Glc/UDP-Gal was 3.5. The enzyme in crude as well as in purified samples was unusually stable and was not inactivated even on incubation at 46 degrees C for several hours.