Kumar V, Heinemann F S, Ozols J
Department of Biochemistry, University of Connecticut Health Center, Farmington 06030.
Anal Biochem. 1994 Jun;219(2):305-8. doi: 10.1006/abio.1994.1270.
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.
寡糖基转移酶催化寡糖从脂质多萜醇转移至新生多肽链上的天冬酰胺受体位点。我们基于反应底物和产物在去污剂溶液中的特定分布开发了一种针对该酶的检测方法。合成了GlcNAc-[³H]GlcNAc-PP-多萜醇用作碳水化合物供体。市售的肽苯甲酰-天冬酰胺-亮氨酸-苏氨酸-酰胺用作寡糖基转移酶的聚糖受体底物。在Triton X-100存在的情况下，GlcNAc-[³H]GlcNAc-PP-多萜醇分配到去污剂微团中，而糖基化的受体肽则分配到微团间的水相中。通过超滤实现了GlcNAc-[³H]GlcNAc-PP-多萜醇与糖肽的分离。利用这种方法，微粒体制剂中的寡糖基转移酶活性最低仅需1微克蛋白质即可检测。
