PURIFICATION AND CHARACTERIZATION OF RAT-KIDNEY UDP-N-ACETYLGLUCOSAMINE - ALPHA-6-D-MANNOSIDE BETA-1,6-N-ACETYLGLUCOSAMINYLTRANSFERASE

In order to investigate the molecular mechanism of the specific increase of UDP-N-acetylglucosamine:alpha-6-D-mannoside beta-1,6-N-acetylglucosaminyltransferase (GlcNAcT-V, EC 2.4.1.155) activity after viral or oncogenic transformation, we have purified the enzyme from a Triton X-100 extract of rat kidney acetone powder. GlcNAcT-V was purified by sequential affinity chromatography using first UDP-hexanolamine-agarose and then a synthetic oligosaccharide inhibitor-agarose column. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme revealed two major bands at apparent molecular masses of 69 and 75 kDa. The enzyme was recovered in a 26% final yield with a 450,000-fold increase in specific activity to a V(max) of 18.8-mu-mol/(mg.min). Enzyme activity was stabilized and enhanced by the addition of 20% glycerol, 0.5 mg/ml IgG, and 0.2 M NaCl. The optimal ranges of pH and Triton X-100 concentrations for enzyme activity were 6.5-7.0 and 1.0-1.5%, respectively. The divalent cations, Mn2+, Ca2+, and Mg2+, were each found to have a negligible (< 10%) effect on activity; moreover, the enzyme was fully active in the presence of 20 mM EDTA. The K(m) value of the purified enzyme toward a synthetic trisaccharide acceptor was 90-mu-M, and the K(i) value toward a synthetic active site inhibitor was 140-mu-M.