Fischer B E, Thomas K B, Schlokat U, Dorner F
IMMUNO AG, Biomedical Research Center, Uferstrasse. 15, A-2304 Orth/Donau, Austria.
Biochem J. 1998 Apr 15;331 ( Pt 2)(Pt 2):483-8. doi: 10.1042/bj3310483.
Human von Willebrand factor (hp-vWF) is a high-molecular- mass protein found in plasma as a series of multimers. It consists of subunits comprising 2050 amino acids linked by disulphide bonds into multimers of various size ranging in molecular mass up to greater than 10000kDa. Partial proteolysis at position Tyr842-Mer843 of the subunit [Dent et al. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 6306-6310] by a vWF-specific protease [Furlan et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 7503-7507] results in the generation of an N-terminal and a C-terminal fragment and the appearance of hp-vWF triplet bands. It has been suggested [Furlan et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 7503-7507] that (i) the intermediate triplet band of the primary dimer represents a dimer of two C-terminal fragments, (ii) the slower migrating satellite band of the primary dimer represents an asymmetric structure composed of a mature subunit to which one N-terminal and one C-terminal fragment are linked by disulphide bonds, and (iii) the faster migrating satellite band of the primary dimer contains two N-terminal fragments. Here we used recombinant vWF (r-vWF) for structural analysis of hp-vWF multimers. r-vWF exhibited no proteolytic degradation and all multimers contained mature subunits. High-resolution agarose-gel electrophoresis and two-dimensional electrophoresis demonstrated that (i) r-vWF multimers and hp-vWF intermediate triplet bands exhibited identical molecular mass and electrophoretic mobilities, (ii) the faster and slower migrating satellite bands of hp-vWF differ by less than the molecular mass of one subunit from the corresponding intermediate triplet band, and (iii) the triplet bands of hp-vWF are composed of mature and degraded subunits. The results support a structural model of hp-vWF triplet bands according to which the intermediate triplet bands represent multiple numbers of symmetric and/or asymmetric dimers, the slower migrating satellite bands have one extra N-terminal fragment, and the faster migrating satellite band lacks one N-terminal fragment respectively in comparison with the corresponding intermediate triplet band.
人血管性血友病因子（hp-vWF）是一种在血浆中以一系列多聚体形式存在的高分子量蛋白质。它由包含2050个氨基酸的亚基组成，这些亚基通过二硫键连接形成各种大小的多聚体，分子量高达大于10000kDa。血管性血友病因子特异性蛋白酶[Furlan等人（1993年）《美国国家科学院院刊》90, 7503 - 7507]在亚基的Tyr842-Mer843位点进行部分蛋白水解[Dent等人（1990年）《美国国家科学院院刊》87, 6306 - 6310]，导致产生一个N端片段和一个C端片段，并出现hp-vWF三联体条带。有人提出[Furlan等人（1993年）《美国国家科学院院刊》90, 7503 - 7507]：（i）初级二聚体的中间三联体条带代表两个C端片段的二聚体；（ii）初级二聚体迁移较慢的卫星条带代表一种不对称结构，由一个成熟亚基组成，一个N端片段和一个C端片段通过二硫键与之相连；（iii）初级二聚体迁移较快的卫星条带包含两个N端片段。在此，我们使用重组血管性血友病因子（r-vWF）对hp-vWF多聚体进行结构分析。r-vWF未表现出蛋白水解降解，所有多聚体均包含成熟亚基。高分辨率琼脂糖凝胶电泳和二维电泳表明：（i）r-vWF多聚体和hp-vWF中间三联体条带表现出相同的分子量和电泳迁移率；（ii）hp-vWF迁移较快和较慢的卫星条带与相应的中间三联体条带相比，分子量差异小于一个亚基的分子量；（iii）hp-vWF的三联体条带由成熟和降解的亚基组成。这些结果支持了hp-vWF三联体条带的结构模型，根据该模型，中间三联体条带代表多个对称和/或不对称二聚体，迁移较慢的卫星条带比相应的中间三联体条带多一个N端片段，迁移较快的卫星条带比相应的中间三联体条带少一个N端片段。
