Blithe D L, Nisula B C
Endocrinology. 1985 Nov;117(5):2218-28. doi: 10.1210/endo-117-5-2218.
The glycoprotein hormone hCG and a free alpha-subunit are secreted by trophoblastic cells during pregnancy. We have purified the alpha-subunit of hCG and the free alpha-subunit population from pregnancy urine. Dissociation of hCG with 10 M urea at 37 C, followed by chromatography on DEAE-Sephacel, resulted in separation of alpha- from beta-subunit, as hCG alpha does not bind to DEAE in the presence of urea, while beta-subunit does bind. Similar treatment of the free alpha population resulted in fractionation into two populations, a nonbinding fraction of free alpha and a population which was retained by DEAE in the presence of urea (free alpha 2). The three populations, hCG alpha, free alpha, and free alpha 2, were further purified by affinity chromatography using anti-alpha antisera linked to Sepharose. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the preparations showed that hCG alpha consisted of a single component with an apparent mol wt of 22,000, while free alpha and free alpha 2 consisted of multiple components. Radioactive labeling of sialic acid by limited periodate oxidation and NaB[3H]4 reduction resulted in a higher specific activity for free alpha than for hCG alpha, suggesting that free alpha contains more sialic acid per immunoreactive molecule than does alpha dissociated from hCG. [3H]hCG alpha, but not 3H-labeled free alpha, was able to combine with native hCG beta-subunit. The oligosaccharide moieties were released from the different labeled subunits by alkaline hydrolysis and then compared with respect to Concanavalin A (ConA)-binding and DEAE-binding properties. Most of the oligosaccharides from dissociated hCG alpha bound to ConA-Sepharose (72%), while less material from free alpha (40%) and even less from free alpha 2 (25%) were capable of binding to ConA. DEAE chromatography of the oligosaccharides suggested that hCG alpha contained primarily monosialylated forms (greater than 60%), while free alpha and alpha 2 contained primarily (greater than 70%) di- and trisialylated forms. Thus, the ConA and DEAE binding data indicated that the oligosaccharide contents of free alpha and free alpha 2 were quite different from that of hCG alpha. These results also suggest that some of the oligosaccharide structures contained on hCG alpha and most of those on free alpha and free alpha 2 differ substantially from the structures that have been previously described.
在怀孕期间，滋养层细胞会分泌糖蛋白激素人绒毛膜促性腺激素（hCG）和游离α亚基。我们从孕妇尿液中纯化了hCG的α亚基和游离α亚基群体。在37℃下用10M尿素使hCG解离，随后在DEAE - 琼脂糖凝胶上进行层析，导致α亚基与β亚基分离，因为在尿素存在下hCGα不与DEAE结合，而β亚基则结合。对游离α群体进行类似处理，结果分成两个群体，一个是游离α的非结合部分，另一个是在尿素存在下被DEAE保留的群体（游离α2）。通过使用与琼脂糖偶联的抗α抗血清进行亲和层析，进一步纯化了hCGα、游离α和游离α2这三个群体。制备物的十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳显示，hCGα由单一成分组成，表观分子量为22,000，而游离α和游离α2由多种成分组成。通过有限的高碘酸盐氧化和NaB[3H]4还原对唾液酸进行放射性标记，结果表明游离α的比活性高于hCGα，这表明每个免疫反应性分子中游离α含有的唾液酸比从hCG解离的α更多。[3H]hCGα能够与天然hCGβ亚基结合，但3H标记的游离α则不能。通过碱性水解从不同标记的亚基中释放寡糖部分，然后比较它们与伴刀豆球蛋白A（ConA）结合和DEAE结合的特性。从解离的hCGα中得到的大多数寡糖与ConA - 琼脂糖凝胶结合（72%），而游离α中较少的物质（40%）以及游离α2中更少的物质（25%）能够与ConA结合。寡糖的DEAE层析表明，hCGα主要含有单唾液酸化形式（大于60%），而游离α和α2主要含有（大于70%）二唾液酸化和三唾液酸化形式。因此，ConA和DEAE结合数据表明，游离α和游离α2的寡糖含量与hCGα有很大不同。这些结果还表明，hCGα上含有的一些寡糖结构以及游离α和游离α2上的大多数寡糖结构与先前描述的结构有很大差异。
