N ‐Glycosylation of Yeast Proteins

The enzyme transferring the oligosaccharide from Dol PP ‐(GlcNAc) 2 (Man) 9 (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 Dol PP ‐[ 14 C]saccharidcs 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 Mn 2+ is optimal; Mg 2+ at a concentration of 10 mM yields only one third the activity observed with Mn 2‐ . 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 Dol PP ‐(GlcNAc) 2 (Man) 9 and from DOl PP ‐GlcNAc. 4 The K m value for Dol PP ‐(GlcNAc) 2 (Man) 9 (Glc) 3 of 0.5 μM does not differ significantly from that for Dol PP ‐(GlcNAc) 2 of 1.2 μM. A broad pH‐optimum for the reaction with both substrates was found between 6.5 and 7.7. 5 However, a clear difference in K m values for the hexapeptide was observed with different dolichol‐linked sugar derivatives. With DOl PP ‐(GlcNAc) 2 a peptide concentration of 0.6 mM resulted in half‐maximal transfer rate, whereas 0.05 mM peptide were sufficient with (GlcNAc) 2 (Man) 9 (Glc) 3 as donor.