<em>N</em>-Glycosylation of Yeast Proteins.

The enzyme transferring the oligosaccharide from DolPP-(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 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 of 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 10 mM yields one third the activity observed with Mn²⁺. 3. The enzyme transfers besides doluchyl-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-(GlcNA)₂(Man)₉ and from DolPP-GlcNAc. The K_m value for DolPP-(GlcNAc)₂(Man)₉(Glc)₃ of 0.5 µm does not differ significantly from that for DolPP-(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 hexpeptide was observed with different dolichol-linked sugar derivatives. With DolPP-(GlcNAc)₂ a peptide concentration of 0.6 mM resulted in half-maximal transfer rate, whereas 0.05 mM peptide were dufficient with DolPP-(GlcNAc)₂(Man)₉(Glc)₃ as donor. [ABSTRACT FROM AUTHOR]