Purification and characterization of rat kidney UDP-N-acetylglucosamine: alpha-6-D-mannoside beta-1,6-N-acetylglucosaminyltransferase

J Biol Chem. 1992 Feb 15;267(5):2920-7.


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 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 Vmax of 18.8 mumols/(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 (less than 10%) effect on activity; moreover, the enzyme was fully active in the presence of 20 mM EDTA. The Km value of the purified enzyme toward a synthetic trisaccharide acceptor was 90 microM, and the Ki value toward a synthetic active site inhibitor was 140 microM.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Cations, Divalent
  • Chromatography, Affinity / methods
  • Electrophoresis, Polyacrylamide Gel
  • Glucosyltransferases / isolation & purification*
  • Glucosyltransferases / metabolism
  • Kidney / enzymology*
  • Kinetics
  • Macromolecular Substances
  • Molecular Weight
  • N-Acetylglucosaminyltransferases*
  • Osmolar Concentration
  • Rats


  • Cations, Divalent
  • Macromolecular Substances
  • Glucosyltransferases
  • N-Acetylglucosaminyltransferases
  • alpha-1,6-mannosylglycoprotein beta 1,6-N-acetylglucosaminyltransferase