Codington J F, Deak M R, Frim D M, Jeanloz R W
Arch Biochem Biophys. 1986 Nov 15;251(1):47-54. doi: 10.1016/0003-9861(86)90049-4.
Mannose-labeled epiglycanin was prepared by incubation of TA3-Ha ascites cells with [2-3H]mannose, removal of the epiglycanin by incubation of viable cells with L-1-p-tosylamino-2-phenylethyl chloromethyl ketone-trypsin, and isolation of the large epiglycanin glycopeptides by gel filtration. Purification of epiglycanin glycopeptides was performed by wheat germ agglutinin affinity chromatography. Extensive incubation of epiglycanin with Pronase, followed by passage through a calibrated column of Bio-Gel P-4 (Column P-4), gave three fractions. The fraction of lowest apparent molecular weight, about 5000, upon incubation with a purified extract from F. meningosepticum containing an N-glycosyl hydrolase and an endo-N-acetyl-beta-D-glucosaminidase (T.H. Plummer et. al. (1984) J. Biol. Chem. 259, 10700-10704) and passage through Column P-4 gave a peak of radioactivity at apparent Mr 3000. Incubation of nonlabeled epiglycanin under similar conditions with the same enzyme preparation followed by passage through Column P-4, gave two peaks, based upon total mannose content. One of these, partially deglycosylated epiglycanin, was present in the void volume. Its composition indicated that approximately 80% of the mannose content of epiglycanin had been removed by the enzyme treatment, whereas no change was noted in the proportion of the other carbohydrate components. The effluent volume of the second peak coincided precisely with the peak obtained from the Pronase-cleaved fraction. Its composition and apparent Mr were consistent with those of an N-lactosamine-type chain with four antennae, Man3Gal4GlcNAc5NeuAc2-3.
将TA3-Ha腹水细胞与[2-³H]甘露糖一起孵育，用L-1-对甲苯磺酰胺基-2-苯乙基氯甲基酮-胰蛋白酶孵育活细胞以去除表皮糖蛋白，然后通过凝胶过滤分离大的表皮糖蛋白糖肽。表皮糖蛋白糖肽的纯化通过麦胚凝集素亲和色谱法进行。表皮糖蛋白与链霉蛋白酶充分孵育，然后通过校准的Bio-Gel P-4柱（柱P-4），得到三个级分。将最低表观分子量约为5000的级分与含有N-糖基水解酶和内切-N-乙酰-β-D-氨基葡萄糖苷酶的脑膜炎败血伊丽莎白金菌纯化提取物一起孵育（T.H. Plummer等人，(1984)《生物化学杂志》259, 10700 - 10704），然后通过柱P-4，在表观分子量为3000处出现放射性峰值。在类似条件下用相同的酶制剂孵育未标记的表皮糖蛋白，然后通过柱P-4，根据总甘露糖含量得到两个峰值。其中之一是部分去糖基化的表皮糖蛋白，存在于空体积中。其组成表明，表皮糖蛋白中约80%的甘露糖含量已被酶处理去除，而其他碳水化合物成分的比例没有变化。第二个峰值的流出体积与链霉蛋白酶裂解级分得到的峰值完全一致。其组成和表观分子量与具有四个触角的N-乳糖胺型链Man3Gal4GlcNAc5NeuAc2 - 3一致。
