title: Characterization and partial purification of a novel enzymatic activity. UDP-GlcNAc:Ser-protein N-acetylglucosamine-1-phosphotransferase from the cellular slime mold Dictyostelium discoideum.
abstract: An enzymatic activity that transfers N-acetylglucosamine-1-phosphate residues from UDP-GlcNAc to serine units in proteins (UDP-GlcNAc:Ser-protein N-acetylglucosamine-1-phosphotransferase) was detected in membranes of the cellular slime mold Dictyostelium discoideum. The enzyme was partially purified by affinity chromatography in concanavalin A-Sepharose and ion exchange chromatography in a Mono Q column. The enzyme showed an absolute requirement for bivalent cations, Mn2+ being more effective than Mg2+. It had a broad optimum pH value (6.5-9.0). The Km for UDP-GlcNAc was 18 microM. In cell free assays it used apomucin and native or 8 M urea-denatured thyroglobulin but neither bovine serum albumin nor native or denatured uteroferrin as exogenous acceptors. Analysis of proteins isolated from cells grown in the presence of [32P]phosphate and from the culture medium showed that the majority of proteins bearing the structure Glc-NAc-1-P-Ser were secreted. In equilibrium density centrifugations of microsomes, the enzyme appeared in membranes having lighter densities than the enzyme that phosphorylates high mannose-type oligosaccharides. This showed that the activity that phosphorylates serine residues in proteins (UDP-GlcNAc:Ser-protein N-acetylglucosamine-1-phosphotransferase) is different from that phosphorylating protein-linked high mannose-type oligosaccharides (UDP-GlcNAc:glycoprotein N-acetylglucosamine-1-phosphotransferase).
authors.affiliation: Instituto de Investigaciones Bioquímicas Fundación Campomar, Buenos Aires, Argentina.
authors.forename: S
authors.initials: S
authors.lastname: Merello
authors.forename: A J
authors.initials: AJ
authors.lastname: Parodi
authors.forename: R
authors.initials: R
authors.lastname: Couso
mesh_terms: D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D004023:Dictyostelium / Q000201:enzymology*; D006031:Glycosylation; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D013347:Subcellular Fractions / Q000201:enzymology; D013379:Substrate Specificity; D017855:Transferases (Other Substituted Phosphate Groups) / Q000302:isolation & purification* / Q000378:metabolism*; D014537:Uridine Diphosphate N-Acetylglucosamine / Q000378:metabolism
chemical_list: D008345:Manganese; D014537:Uridine Diphosphate N-Acetylglucosamine; D017855:Transferases (Other Substituted Phosphate Groups); C092951:UDP-GlcNAc - Ser-protein N-acetylglucosamine-1-phosphotransferase; D008274:Magnesium