Miller R P, Farley R A
Department of Physiology and Biophysics, USC School of Medicine, Los Angeles 90033.
Biochim Biophys Acta. 1988 Apr 28;954(1):50-7. doi: 10.1016/0167-4838(88)90054-4.
The beta-subunit of dog kidney (Na+ + K+)-ATPase is a sialoglycoprotein and contains three potential N-glycosylation sites. In this study, the oligosaccharide chains of purified dog kidney beta-subunit were labeled with tritium by oxidation with sodium periodate or galactose oxidase followed by NaB3H4 reduction. The beta-subunit was extensively digested by trypsin and the radioactive peptides were purified by HPLC. The enzyme, glycopeptidase A, which catalyzes the removal of N-linked oligosaccharide chains and the conversion of the glycosylated Asn residue to Asp, was used to demonstrate that a number of purified beta-subunit tryptic peptides were glycosylated. Amino-acid analysis of these beta-subunit peptides following glycopeptidase-A treatment revealed the expected Asn to Asp conversion for Asn-157, Asn-192 and Asn-264, demonstrating that all three potential N-glycosylation sites of the dog kidney beta-subunit are glycosylated. In addition, amino-acid sequence data suggest that a disulfide bond exists between Cys-158 and Cys-174.
犬肾（Na⁺ + K⁺）-ATP酶的β亚基是一种唾液酸糖蛋白，含有三个潜在的N-糖基化位点。在本研究中，纯化的犬肾β亚基的寡糖链先用高碘酸钠或半乳糖氧化酶氧化，再用NaB₃H₄还原，用氚进行标记。β亚基用胰蛋白酶进行广泛消化，放射性肽通过高效液相色谱法纯化。使用催化去除N-连接寡糖链并将糖基化的天冬酰胺残基转化为天冬氨酸的糖肽酶A来证明许多纯化的β亚基胰蛋白酶肽是糖基化的。糖肽酶A处理后对这些β亚基肽进行氨基酸分析，结果显示天冬酰胺-157、天冬酰胺-192和天冬酰胺-264出现了预期的从天冬酰胺到天冬氨酸的转化，表明犬肾β亚基的所有三个潜在N-糖基化位点均被糖基化。此外，氨基酸序列数据表明半胱氨酸-158和半胱氨酸-174之间存在二硫键。
