Microassay for oligosaccharyltransferase: separation of reaction components by partitioning in detergent solution followed by ultrafiltration.
Oligosaccharyltransferase catalyzes the transfer of oligosaccharide from a lipid dolichol to asparagine acceptor sites on nascent polypeptides. We have developed an assay for this enzyme which is based on the specific distribution of the substrate and product of the reaction in detergent solution. GlcNAc-[3H]GlcNAc-PP-Dol was synthesized for use as a carbohydrate donor. Benzoyl-Asn-Leu-Thr-amide, a commercially available peptide, was used as the oligosaccharyltransferase glycan acceptor substrate. In the presence of Triton X-100, GlcNAc-[3H]GlcNAc-PP-Dol partitions into detergent micelles while glycosylated acceptor peptide partitions into the intermicellar aqueous compartment. Separation of GlcNAc-[3H]GlcNAc-PP-Dol and glycopeptide was achieved by ultrafiltration. With this method oligosaccharyltransferase activity in microsomal preparations could be measured with as little as 1 microgram of protein.
Kumar
V
V
Department of Biochemistry, University of Connecticut Health Center, Farmington 06030.
Heinemann
F S
FS
Ozols
J
J
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001345:Autoradiography / Q000379:methods; D002240:Carbohydrate Sequence; D002645:Chickens; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003902:Detergents; D005260:Female; D006602:Hexosyltransferases; D008565:Membrane Proteins; D008832:Microchemistry / Q000379:methods; D008861:Microsomes / Q000201:enzymology; D008969:Molecular Sequence Data; D010057:Oviducts / Q000201:enzymology; D011104:Polyisoprenyl Phosphate Oligosaccharides / Q000138:chemical synthesis / Q000378:metabolism; D013379:Substrate Specificity; D014166:Transferases / Q000032:analysis* / Q000302:isolation & purification / Q000378:metabolism; D014316:Tritium; D014462:Ultrafiltration / Q000379:methods
D000596:Amino Acids; D003902:Detergents; D008565:Membrane Proteins; D011104:Polyisoprenyl Phosphate Oligosaccharides; D014316:Tritium; C016418:dolichyl-diphosphate-di-N-acetylchitobiose; D014166:Transferases; D006602:Hexosyltransferases; C025620:dolichyl-diphosphooligosaccharide - protein glycotransferase