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 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.
Shoreibah
M G
MG
Department of Biochemistry, University of Georgia, Athens 30602.
Hindsgaul
O
O
Pierce
M
M
D000818:Animals; D002413:Cations, Divalent; D002846:Chromatography, Affinity / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D005964:Glucosyltransferases / Q000302:isolation & purification* / Q000378:metabolism; D007668:Kidney / Q000201:enzymology*; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D017351:N-Acetylglucosaminyltransferases; D009994:Osmolar Concentration; D051381:Rats
D002413:Cations, Divalent; D046911:Macromolecular Substances; D005964:Glucosyltransferases; D017351:N-Acetylglucosaminyltransferases; C052254:alpha-1,6-mannosylglycoprotein beta 1,6-N-acetylglucosaminyltransferase