Oligosaccharyltransferase transfers a preformed oligosaccharide from a dolichol carrier molecule to specific asparaginyl residues of proteins synthesized in the endoplasmic reticulum. We have isolated a protein complex with this activity from chicken liver microsomes with 850 fold purification. The purification procedure involved removal of peripheral and lumenal proteins, solubilization of the membranes by non-ionic detergent and glycerol gradient centrifugation. The complex was purified further by ion-exchange and gel filtration chromatography. SDS-PAGE analysis of the final preparation revealed 3 major protein bands, two bands with an approximate molecular weight of 65-kDa and one band of approximately 50-kDa. Endoglycosidase H digestion of the purified subunits indicated the presence of carbohydrate on the 65-I subunit. No carbohydrate was detected in the 65-II subunit or the 50-kDa subunit. Amino acid sequence analysis of the intact protein subunits and internal peptides generated by cynogen bromide digestion, identified the 65-kDa subunits as ribophorin I and II. The 50-kDa subunit has 25% homology with a yeast membrane protein (Wbplp) which is essential for oligosaccharyltransferase activity in Saccharomyces cerevisiae.
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