Glycosylation of nuclear and cytoplasmic proteins. Purification and characterization of a uridine diphospho-N-acetylglucosamine:polypeptide beta-N-acetylglucosaminyltransferase.
Using a combination of conventional and affinity chromatographic techniques, we have purified a uridine diphospho-N-acetylglucosamine:polypeptide beta-N-acetylglucosaminyltransferase (O-GlcNAc transferase) over 30,000-fold from rat liver cytosol. The transferase is soluble and very large, migrating with an apparent molecular weight of 340,000 on molecular sieve chromatography. Analysis of the purified enzyme on sodium dodecyl sulfate-polyacrylamide gel electrophoresis reveals two protein species migrating at 110 (alpha subunit) and 78 (beta subunit) kDa in approximately a two-to-one ratio. Thus, the enzyme likely exists as a heterotrimer complex with two subunits of 110 kDa and one of 78 kDa (alpha 2 beta). The alpha subunit appears to contain the enzyme's active site since it is selectively radiolabeled by a specific photoaffinity probe (4-[beta-32P]thiouridine diphosphate). Photoinactivation and photolabeling of the enzyme are dependent on time and long wavelength ultraviolet light. Photolabeling of the alpha subunit is specifically blocked by UDP. The enzyme has an extremely high affinity for UDP-GlcNAc (Km = 545 nM). This unusually high affinity for the sugar nucleotide donor probably provides the enzyme an advantage over the nucleotide transporters in the endoplasmic reticulum and Golgi apparatus which compete for available cytoplasmic UDP-GlcNAc. The multimeric state and large size of the O-GlcNAc transferase imply that its activity may be highly regulated within the cell.
Haltiwanger
R S
RS
Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
Blomberg
M A
MA
Hart
G W
GW
D000345:Affinity Labels; D000595:Amino Acid Sequence; D000818:Animals; D002850:Chromatography, Gel; D003593:Cytoplasm / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005964:Glucosyltransferases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D006031:Glycosylation; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D017351:N-Acetylglucosaminyltransferases; D009687:Nuclear Proteins / Q000378:metabolism*; D009711:Nucleotides / Q000494:pharmacology; D010777:Photochemistry; D051381:Rats; D013379:Substrate Specificity
D000345:Affinity Labels; D009687:Nuclear Proteins; D009711:Nucleotides; D005964:Glucosyltransferases; D017351:N-Acetylglucosaminyltransferases; C063057:UDP-N-acetylglucosamine-peptide beta-N-acetylglucosaminyltransferase