PURIFICATION AND CHARACTERIZATION OF UDP-D-GALACTOSE 4-EPIMERASE FROMTHE RED ALGA GALDIERIA-SULPHURARIA

UDP-D-galactose 4-epimerase of the unicellular red alga Galdieria sulp huraria has been purified to apparent electrophoretic homogeneity by c hromatography on DEAE-Fractogel, hydroxylapatite and by affinity chrom atography on Dyematrex Orange. The holoenzyme is a homodimer with an a pparent 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 polyacryl amide gel electrophoresis. The 4-epimerase from G. sulphuraria does no t 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 appa rent equilibrium constant for UDP-Glc/UDP-Gal was 3.5. The enzyme in c rude as well as in purified samples was unusually stable and was not i nactivated even on incubation at 46 degrees C for several hours.