Purification and characterization of hepatic oligosaccharyltransferase.
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.
Department of Biochemistry, University of Connecticut Health Center, Farmington 06030, USA.
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Kumar
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Heinemann
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Ozols
D000595:Amino Acid Sequence; D000818:Animals; D002499:Centrifugation, Density Gradient; D002645:Chickens; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003488:Cyanogen Bromide; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006602:Hexosyltransferases; D007700:Kinetics; D046911:Macromolecular Substances; D008565:Membrane Proteins; D008862:Microsomes, Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry; D010449:Peptide Mapping; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D017386:Sequence Homology, Amino Acid; D014166:Transferases / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism
D046911:Macromolecular Substances; D008565:Membrane Proteins; D010446:Peptide Fragments; D014166:Transferases; D006602:Hexosyltransferases; C025620:dolichyl-diphosphooligosaccharide - protein glycotransferase; D003488:Cyanogen Bromide