Cantor A B, Kornfeld S
Department of Internal Medicine, Washington University School of Medicine, St. Louis, Missouri 63110.
Anal Biochem. 1992 Sep;205(2):220-6. doi: 10.1016/0003-2697(92)90427-9.
We have developed an efficient method for labeling the Asn-linked oligosaccharides of recombinant glycoproteins synthesized in Xenopus laevis oocytes. By coinjecting GDP-[3,4-(3)H]mannose with mRNA for human cathepsin D, it was possible to incorporate as much as 1800 cpm per oocyte into each of the two Asn-linked oligosaccharides of this glycoprotein. Overall, about 50% of the microinjected GDP-[3,4-(3)H]mannose was incorporated into Asn-linked oligosaccharides, a 10-fold greater value than that obtained when [2-(3)H]mannose was microinjected. Less than 10% of the injected GDP-[3,4-(3)H]mannose was metabolized to water or converted to amino acids. This technique should facilitate studies of Asn-linked oligosaccharide biosynthesis, processing, and structure in recombinant proteins synthesized in Xenopus oocytes.
我们已经开发出一种高效的方法，用于标记在非洲爪蟾卵母细胞中合成的重组糖蛋白的天冬酰胺连接寡糖。通过将GDP-[3,4-(3)H]甘露糖与人组织蛋白酶D的mRNA共同注射，有可能将每个卵母细胞多达1800 cpm的放射性掺入到这种糖蛋白的两个天冬酰胺连接寡糖中。总体而言，约50%的显微注射的GDP-[3,4-(3)H]甘露糖被掺入到天冬酰胺连接寡糖中，这一数值比显微注射[2-(3)H]甘露糖时获得的数值大10倍。注射的GDP-[3,4-(3)H]甘露糖中不到10%被代谢为水或转化为氨基酸。这项技术应有助于对非洲爪蟾卵母细胞中合成的重组蛋白中天冬酰胺连接寡糖的生物合成、加工和结构进行研究。
