Human plasma uridine diphosphate galactose-glycoprotein galactosyltransfertase. Purification, properties and kinetics of the enzyme-catalysed reaction.
The soluble galactosyltransferase of human plasma catalysed the transfer of galactose from UDP-galactose to high- and low-molecular-weight derivatives of N-acetylglucosamine, forming a beta-1-4 linkage. The enzyme was purified by using (NH4)2SO4 precipitation and affinity chromatography on an alpha-lactalbumin-Sepharose column. The galactosyltransferase was maximally bound to this column in the presence of N-acetylglucosamine, and the enzyme was eluted by omitting the amino sugar from the developing buffer. The molecular weight of the enzyme was estimated to be 85000 by gel filtration. The assay conditions for optimum enzymic activity was 30 degrees C and pH7.5. Mn2+ ion was found to be an absolute requirement for transferase activity. The Km for Mn2+ was 0.4 mM and that for the substrate, UDP-galactose, was 0.024 mM. The Km for the acceptors was 0.21 mM for alpha1-acid glycoprotein and 3.9 mM for N-acetylglucosamine. In the presence of alpha-lactalbumin, glucose became a good acceptor for the enzyme and had a Km value of 2.9 mM. Results of the kinetic study indicated that the free enzyme reacts with Mn2+ under conditions of thermodynamic equilibrium, and the other substrates are added sequentially.
D002854:Chromatography, Paper; D005700:Galactosyltransferases / Q000097:blood*; D006801:Humans; D007700:Kinetics; D008345:Manganese / Q000494:pharmacology; D008970:Molecular Weight; D013379:Substrate Specificity; D006022:beta-N-Acetylglucosaminylglycopeptide beta-1,4-Galactosyltransferase / Q000097:blood* / Q000302:isolation & purification
D008345:Manganese; D005700:Galactosyltransferases; D006022:beta-N-Acetylglucosaminylglycopeptide beta-1,4-Galactosyltransferase