Purification and characterization of human lymphoblast N-acetylglucosamine-1-phosphotransferase
- 1 April 1992
- journal article
- research article
- Published by Oxford University Press (OUP) in Glycobiology
- Vol. 2 (2) , 119-125
- https://doi.org/10.1093/glycob/2.2.119
Abstract
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 μM for the donor UDP-N-acetylglucosamine and of 117 mM for the artificial acceptor α-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 ∼1000 kDa, which is analogous to that reported for the partially purified enzyme from rat liver (Waheed et al., 1982).Keywords
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