Active site-directed photoaffinity labeling and partial characterization of oligosaccharyltransferase.
Oligosaccharyltransferase, the enzyme that catalyzes the transfer of the oligosaccharide chain of dolichol-P-P-GlcNAc2Man9Glc3 to asparagine residues in -Asn-X-Thr/Ser- sites within polypeptides, has been radiolabeled using a photoactivatable azido tripeptide acceptor, N alpha-[3H]Ac-Asn-Lys(N epsilon-p-azidobenzoyl)-Thr-NH2. As determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the molecular mass of the oligosaccharyltransferase polypeptide from hen oviduct microsomes is 60 kDa. Radiolabeling of the 60-kDa polypeptide was completely dependent upon photolysis of hen oviduct endoplasmic reticulum preparations in the presence of the azido peptide and Mn2+, which is required for enzymatic activity. Labeling of the enzyme was not inhibited in the presence of a 10-fold excess of the nonacceptor peptides, unacetylated Asn-Lys(N epsilon-p-azidobenzoyl)-Thr-NH2 or unacetylated Asn-Leu-Thr-NH2, whereas it was completely abolished by the presence of a 10-fold excess of the competing acceptor peptide, N alpha-Bz-Asn-Leu-Thr-NH2. Thermal inactivation of oligosaccharyltransferase was achieved by heating endoplasmic reticulum preparations to 60 degrees C. This loss of enzyme activity at 60 degrees C paralleled a comparable decrease in radiolabeling of the 60-kDa polypeptide, whereas temperatures of 50 degrees C and lower had no effect on either process. Oligosaccharyltransferase itself may be an N-linked glycoprotein, because the 60-kDa radiolabeled polypeptide binds to concanavalin A-agarose and is susceptible to digestion by beta-endohexosaminidase H.
D000345:Affinity Labels; D000818:Animals; D001386:Azides; D001665:Binding Sites; D002645:Chickens; D004721:Endoplasmic Reticulum / Q000201:enzymology; D005260:Female; D006602:Hexosyltransferases; D006358:Hot Temperature; D008565:Membrane Proteins; D009842:Oligopeptides; D010057:Oviducts / Q000201:enzymology; D010777:Photochemistry; D011489:Protein Denaturation; D014166:Transferases / Q000378:metabolism*
D000345:Affinity Labels; D001386:Azides; D008565:Membrane Proteins; D009842:Oligopeptides; C045456:N(alpha)-acetylasparaginyl-lysyl-N(epsilon)-4-azidobenzoyl-threoninamide; D014166:Transferases; D006602:Hexosyltransferases; C025620:dolichyl-diphosphooligosaccharide - protein glycotransferase