Kato Y, Iwase H, Hotta K
J Biochem. 1984 Feb;95(2):455-63. doi: 10.1093/oxfordjournals.jbchem.a134627.
Ovalbumin was extracted with a buffered Triton X-100 solution from oviduct slices incubated with [35S]methionine or [2-3H]mannose and was purified by CM-cellulose and Sephacryl S-200 column chromatography. The labeled ovalbumin was fractionated on a concanavalin A (Con A)/Sepharose column at room temperature. Six fractions were separated: Two unadsorbed fractions, OA and OB; and four adsorbed fractions, OC, OD, OE, and OF. Fractions OA, OB, OC, and OD corresponded to the four fractions prepared from unlabeled ovalbumin as previously reported (Iwase, H. et al. (1981) J. Biol. Chem. 256, 5638-5642). Fractions OE and OF were novel constituents of the labeled ovalbumin, although a small amount of OE was also present in unlabeled ovalbumin. Pulse-chase experiments indicated that both OE and OF behaved as biosynthetic intermediates of the other ovalbumin fractions, namely OA, OB, OC, and OD. Partial structural analyses involving endo-beta-N-acetylglucosaminidase treatment and subsequent Bio-Gel P-4 chromatography showed that both OE and OF were composed of components bearing high mannose-type sugar chains which consisted of Man9GlcNAc2, Man8GlcNAc2, or Man7GlcNAc2. We failed to distinguish OF from OE on the basis of their carbohydrate chains. However, on SDS slab gel electrophoresis, the OF band migrated slower than the OE band.
用含缓冲液的Triton X-100溶液从与[35S]甲硫氨酸或[2-3H]甘露糖一起孵育的输卵管切片中提取卵清蛋白，然后通过CM-纤维素和Sephacryl S-200柱色谱法进行纯化。将标记的卵清蛋白在室温下在伴刀豆球蛋白A（Con A）/琼脂糖柱上进行分级分离。分离出六个级分：两个未吸附级分，OA和OB；以及四个吸附级分，OC、OD、OE和OF。级分OA、OB、OC和OD对应于先前报道的从未标记卵清蛋白制备的四个级分（Iwase, H.等人，(1981) J. Biol. Chem. 256, 5638 - 5642）。级分OE和OF是标记卵清蛋白的新成分，尽管在未标记的卵清蛋白中也存在少量的OE。脉冲追踪实验表明，OE和OF均表现为其他卵清蛋白级分（即OA、OB、OC和OD）的生物合成中间体。涉及内切β-N-乙酰葡糖胺酶处理及随后的Bio-Gel P-4色谱分析的部分结构分析表明，OE和OF均由带有高甘露糖型糖链的成分组成，这些糖链由Man9GlcNAc2、Man8GlcNAc2或Man7GlcNAc2组成。我们无法根据它们的糖链区分OF和OE。然而，在SDS平板凝胶电泳上，OF条带的迁移速度比OE条带慢。
