PMID: 7018901May 1, 1981Paper

N-Glycosylation of yeast proteins. Characterization of the solubilized oligosaccharyl transferase

European Journal of Biochemistry
C B SharmaW Tanner

Abstract

The enzyme transferring the oligosaccharide from DolPP-(GlcNAc)2(Glc)3 to asparagine residues of glycoproteins has been solubilized from yeast membranes by extraction with detergents. Enzyme activity was tested by measuring transfer of the glycosyl moiety from DolPP-[14C]saccharides to the hexapeptide Tyr-Asn-Leu-Thr-Ser-Val. The rate of transfer was linear for 20 min, with about 40% of dolichyl-diphosphate-bound radioactivity transferred to the peptide. The solubilized enzyme has been characterized as follows: 1. The enzyme is most efficiently solubilized (60% of the membrane-associated activity) by 0.5% Nonidet P40 at a protein/detergent ratio of 2. Octylglucoside solubilizes one third of the activity, but strongly inhibits the reaction if present in the test at a concentration of 1%. 2. Divalent cations are absolutely required. 1 mM Mn2+ is optimal; Mg2+ at a concentration of 10mM yields only one third the activity observed with Mn2+. 3. The enzyme transfers besides dolichyl-diphosphate-bound (GlcNAc)2(Man)9(Glc)3 also (GlcNAc)2(Man)1 and (GlcNAc)2; the rate decreases in this order. No transfer is observed from DolPP-(GlcNAc)2(Man)9 and from DolPP-GlcNAc. 4. The Km value for DolPP-(GlcNAc)2(Man)9(Glc)3 of 0.5 microM does not...Continue Reading

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Citations

Apr 5, 1996·Biotechnology and Bioengineering·M ShelikoffG Stephanopoulos
May 27, 2011·Journal of Inherited Metabolic Disease·Elisabeth MohorkoMarkus Aebi
Mar 1, 1989·Trends in Genetics : TIG·R J DeshaiesP C Böhni
Dec 1, 1995·Bioorganic & Medicinal Chemistry·B Imperiali, T L Hendrickson
Apr 30, 2013·Nature Chemical Biology·Angelyn LarkinBarbara Imperiali
Jun 7, 2006·Proceedings of the National Academy of Sciences of the United States of America·Manasi ChavanHuilin Li
Mar 1, 1982·Proceedings of the National Academy of Sciences of the United States of America·A Haselbeck, W Tanner
Dec 1, 1990·Proceedings of the National Academy of Sciences of the United States of America·C H PedemonteJ H Kaplan
Jun 21, 1994·Proceedings of the National Academy of Sciences of the United States of America·I StagljarM Aebi
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Sep 3, 2003·Bioscience, Biotechnology, and Biochemistry·Yuko NakaharaYoshiaki Nakahara
Apr 21, 2001·Biotechnology Progress·R GonzalezJ A Asenjo
Feb 6, 2013·The Journal of Biological Chemistry·Sabina GerberKaspar P Locher
Apr 16, 2013·Protein Expression and Purification·Marcie B Jaffee, Barbara Imperiali
Mar 17, 2015·Cellular and Molecular Life Sciences : CMLS·Yoichiro HaradaTadashi Suzuki
Aug 29, 2012·Biochemical and Biophysical Research Communications·Widmar Tanner, Ludwig Lehle
Jul 25, 1995·Biochemistry·T L Hendrickson, B Imperiali
Jun 17, 2011·Nature·Christian LizakKaspar P Locher
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Dec 1, 2005·Glycobiology·Daniel J Kelleher, Reid Gilmore
Apr 4, 2007·The Journal of Cell Biology·Daniel J KelleherReid Gilmore

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