Akama T, Yamada K M, Seno N, Matsumoto I, Kono I, Kashiwagi H, Funaki T, Hayashi M
J Biochem. 1986 Nov;100(5):1343-51. doi: 10.1093/oxfordjournals.jbchem.a121840.
The cell-adhesive glycoprotein vitronectin in human plasma was characterized with a monospecific anti-vitronectin antibody. Vitronectin, a mixture of monomeric 75 and 65 kDa polypeptides, was found to have different ratios of amounts of 75 and 65 kDa polypeptides in immunoblots of sera from various healthy human donors. Two states of vitronectin were previously reported; the open state binds to heparin, but the cryptic state does not (Hayashi et al. (1985) J. Biochem. 98, 1135-1138). The anti-vitronectin antibody was suggested to react more strongly with the open state of vitronectin than with the cryptic state. To quantitate all vitronectin regardless of its state, an enzyme-linked immunosorbent assay of vitronectin was developed based on prior boiling of vitronectin-containing samples in 2% (w/v) sodium dodecyl sulfate and 40 mM dithiothreitol to destroy conformational differences. About 12-20% of the vitronectin molecules in plasma were found to bind to heparin-Sepharose under physiological conditions. Vitronectin in plasma bound 30-fold more efficiently to heparin immobilized by amino groups than by carboxyl groups. Its affinity for heparin was higher than for chondroitin sulfate A or C, or dermatan sulfate. Vitronectin was also found to contain covalently-linked small polypeptides of 15 and 13 kDa. These light chains seemed to be disulfide-bonded to the 65 kDa polypeptide, and might be endogenously derived from nicks in the carboxy-terminal portion of the 75 kDa polypeptide in plasma.
利用一种单特异性抗玻连蛋白抗体对人血浆中的细胞黏附糖蛋白玻连蛋白进行了表征。玻连蛋白是由75 kDa和65 kDa单体多肽组成的混合物，发现在来自不同健康人类供体的血清免疫印迹中，75 kDa和65 kDa多肽的含量比例不同。先前报道了玻连蛋白的两种状态；开放状态可与肝素结合，而隐蔽状态则不能（Hayashi等人，（1985年）《生物化学杂志》98卷，1135 - 1138页）。据推测，抗玻连蛋白抗体与玻连蛋白开放状态的反应比与隐蔽状态的反应更强。为了对所有状态的玻连蛋白进行定量，基于将含玻连蛋白的样品在2%（w/v）十二烷基硫酸钠和40 mM二硫苏糖醇中预先煮沸以消除构象差异，开发了一种玻连蛋白的酶联免疫吸附测定法。发现在生理条件下，血浆中约12 - 20%的玻连蛋白分子可与肝素 - 琼脂糖结合。血浆中的玻连蛋白与通过氨基固定的肝素结合的效率比与通过羧基固定的肝素高30倍。它对肝素的亲和力高于对硫酸软骨素A或C或硫酸皮肤素的亲和力。还发现玻连蛋白含有共价连接的15 kDa和13 kDa小多肽。这些轻链似乎通过二硫键与65 kDa多肽相连，可能内源性地源自血浆中75 kDa多肽羧基末端部分的缺口。
