Purification and characterization of human lymphoblast N-acetylglucosamine-1-phosphotransferase.
N-Acetylglucosamine-1-phosphotransferase (GlcNAcPTase) was solubilized with 2% Tergitol NP-10 from cultured human lymphoblast cells and purified 3840-fold with 14% recovery using lentil lectin-Sepharose 4B, DEAE-Sephacel and Sephacryl S-400 chromatographies. The partially purified enzyme requires the non-ionic detergent Tergitol NP-10 and a divalent cation, Mn2+ or Mg2+, for its activity and exhibits an optimal pH at 7.2-7.5 in Tris-maleate buffer. Kinetic studies demonstrated an apparent Km of 24 microM for the donor UDP-N-acetylglucosamine and of 117 mM for the artificial acceptor alpha-methylmannoside. The GlcNAcPTase is inhibited by UDP and UDP-glucose, and by negatively charged phospholipids including phosphatidylserine, phosphatidylglycerol and phosphatidic acid. The apparent mol. wt of the human lymphoblast GlcNAcPTase is approximately 1000 kDa, which is analogous to that reported for the partially purified enzyme from rat liver (Waheed et al., 1982).
Zhao
K W
KW
Department of Neurosciences, University of California, San Diego School of Medicine, La Jolla 92093.
Yeh
R
R
Miller
A L
AL
D002846:Chromatography, Affinity; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D004795:Enzyme Stability; D006801:Humans; D037102:Lectins; D008213:Lymphocyte Activation; D008214:Lymphocytes / Q000201:enzymology*; D010770:Phosphotransferases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification*; D037121:Plant Lectins; D012995:Solubility; D013379:Substrate Specificity; D017855:Transferases (Other Substituted Phosphate Groups)
D037102:Lectins; D037121:Plant Lectins; C033619:lentil lectin; D010770:Phosphotransferases; D017855:Transferases (Other Substituted Phosphate Groups); C030012:UDP-N-acetylglucosamine-lysosomal-enzyme-N-acetylglucosaminephosphotransferase