Bovine UDP-N-acetylglucosamine:lysosomal-enzyme N-acetylglucosamine-1-phosphotransferase. II. Enzymatic characterization and identification of the catalytic subunit
| Autor: | M, Bao, B J, Elmendorf, J L, Booth, R R, Drake, W M, Canfield |
|---|---|
| Rok vydání: | 1996 |
| Předmět: |
Manganese
Tartrate-Resistant Acid Phosphatase Acid Phosphatase Transferases (Other Substituted Phosphate Groups) Hydrogen-Ion Concentration Methylmannosides Cathepsin D Isoenzymes Uridine Diphosphate Galactose Kinetics Ribonucleases Spectrometry Mass Matrix-Assisted Laser Desorption-Ionization Metalloproteins Animals Calcium Cattle Magnesium Rabbits Phosphorylation |
| Zdroj: | The Journal of biological chemistry. 271(49) |
| ISSN: | 0021-9258 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|