Purification and characterization of a galactose-1-phosphate: UDP-glucose uridyltransferase from the red alga Galdieria sulphuraria.
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 microM, Km(UDP-galactose) = 30 microM, Km(Gal1P) = 400 microM, and Km(UDP-Glc) = 20 microM. 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.
Gross
W
W
Freie Universität Berlin, Institut für Pflanzenphysiologie und Mikrobiologie, Germany.
Schnarrenberger
C
C
D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D000461:Rhodophyta / Q000201:enzymology*; D006599:UDPglucose-Hexose-1-Phosphate Uridylyltransferase / Q000302:isolation & purification* / Q000378:metabolism; D005694:UTP-Hexose-1-Phosphate Uridylyltransferase / Q000302:isolation & purification* / Q000378:metabolism
D005694:UTP-Hexose-1-Phosphate Uridylyltransferase; D006599:UDPglucose-Hexose-1-Phosphate Uridylyltransferase