title: Bovine UDP-N-acetylglucosamine:lysosomal-enzyme N-acetylglucosamine-1-phosphotransferase. II. Enzymatic characterization and identification of the catalytic subunit.
abstract: The kinetic properties of UDP-N-acetylglucosamine:lysosomal-enzyme N-acetylglucosamine-1-phosphotransferase (GlcNAc-phosphotransferase) purified to homogeneity from lactating bovine mammary gland have been investigated. GlcNAc-phosphotransferase transferred GlcNAc 1-phosphate from UDP-GlcNAc to the synthetic acceptor alpha-methylmannoside, generating GlcNAc-1-phospho-6-mannose alpha-methyl, the structure of which was confirmed by mass spectroscopy. GlcNAc-phosphotransferase was active between pH 5.7 and 9.3, with optimal activity between pH 6.6 and 7.5. Activity was strictly dependent on Mg2+ or Mn2+. The Km for Mn2+ was 185 microM. The Km for UDP-GlcNAc was 30 microM, and that for alpha-methylmannoside was 63 mM. The enzyme was competitively inhibited by UDP-Glc, with a Ki of 733 microM. The 166-kDa subunit was identified as the catalytic subunit by photoaffinity labeling with azido-[beta-32P]UDP-Glc. Purified GlcNAc-phosphotransferase utilizes the lysosomal enzyme uteroferrin approximately 163-fold more effectively than the non-lysosomal glycoprotein ribonuclease B. Antibodies to GlcNAc-phosphotransferase blocked the transfer to cathepsin D, but not to alpha-methylmannoside, suggesting that protein-protein interactions are required for the efficient utilization of glycoprotein acceptors. These results indicate that the purified bovine GlcNAc-phosphotransferase retains the specificity for lysosomal enzymes as acceptors previously observed with crude preparations.
authors.affiliation: W. K. Warren Medical Research Institute and the Department of Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104, USA. Bill-canfield@uokhsc.edu
authors.forename: M
authors.initials: M
authors.lastname: Bao
authors.forename: B J
authors.initials: BJ
authors.lastname: Elmendorf
authors.forename: J L
authors.initials: JL
authors.lastname: Booth
authors.forename: R R
authors.initials: RR
authors.lastname: Drake
authors.forename: W M
authors.initials: WM
authors.lastname: Canfield
mesh_terms: D000135:Acid Phosphatase; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002402:Cathepsin D / Q000037:antagonists & inhibitors; D002417:Cattle; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D008345:Manganese / Q000378:metabolism; D008667:Metalloproteins / Q000378:metabolism; D008766:Methylmannosides / Q000494:pharmacology; D010766:Phosphorylation; D011817:Rabbits; D012260:Ribonucleases / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D000071681:Tartrate-Resistant Acid Phosphatase; D017855:Transferases (Other Substituted Phosphate Groups) / Q000737:chemistry* / Q000378:metabolism; D014531:Uridine Diphosphate Galactose / Q000494:pharmacology
chemical_list: D007527:Isoenzymes; D008667:Metalloproteins; D008766:Methylmannosides; D014531:Uridine Diphosphate Galactose; D008345:Manganese; C008466:methylmannoside; D017855:Transferases (Other Substituted Phosphate Groups); C030012:UDP-N-acetylglucosamine-lysosomal-enzyme-N-acetylglucosaminephosphotransferase; D012260:Ribonucleases; C029600:ribonuclease B; D000135:Acid Phosphatase; D000071681:Tartrate-Resistant Acid Phosphatase; D002402:Cathepsin D; D008274:Magnesium; D002118:Calcium