HUMAN VONWILLEBRAND-FACTOR - A MULTIVALENT PROTEIN COMPOSED OF IDENTICAL SUBUNITS
HUMAN VONWILLEBRAND-FACTOR - A MULTIVALENT PROTEIN COMPOSED OF IDENTICAL SUBUNITS
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DOI：
10.1021/bi00359a012
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发表时间：
1986-06-03
期刊：
影响因子：
2.9
通讯作者：
TITANI, K
中科院分区：
文献类型：
--
作者：
CHOPEK, MW;GIRMA, JP;TITANI, K
为了研究血管性血友病因子（vWF）的结构及其与血小板的结合活性，建立了从人因子VIII浓缩物中大规模分离vWF的方法。vWF由许多糖蛋白亚基组成，这些亚基通过二硫键连接在一起形成一系列多聚体。这些多计时器似乎包含偶数个270K的亚基。Mr 140K和Mr 120K的两个次要成分也被鉴定出来，但这些链似乎是由次要的蛋白质水解产生的。vWF中最小的多定时器所含的270K、140K和120K亚基几乎是等量的，而最大的多定时器所含的这两种亚基还不到20%。氨基酸序列分析、氨基酸组成和溴化氰裂解表明，270K亚基是相同的，均为单链多肽，氨基端序列为ser - leu - ser - cys - arg - pro - pro - met - valys，羧基端序列为Glu-Cys-Lys-Ser-Pro-Arg-Lys-Cys-Ser-Lys。多聚体大于5个（即含有超过10个270K亚基）的血小板结合比小于3个（含有少于6个270K亚基）的多聚体高8倍。然而，部分还原的vWF (Mr 500K)，无论是由大分子量还是小分子量多聚体制备，都能使血小板结合与最小的多聚体相似。同样，弹性酶、热溶酶、胰蛋白酶或凝乳胰蛋白酶的部分蛋白水解产生小的“多聚体样”蛋白，其与血小板结合特性类似于部分还原的vWF或最小的多聚体。我们得出结论，人类vWF含有相同的270K亚基，组装成多价结构。通过部分还原或部分蛋白水解产生的基本上是单价蛋白，其血小板结合特性与最小的多聚体相似。多价性很可能是导致生物活性增加的主要因素。
A large-scale method for the isolation of von Willebrand factor (vWF) from human factor VIII concentrates was developed in order to study the structure of this protein and its platelet binding activity. vWF is composed of a number of glycoprotein subunits that are linked together by disulfide bonds to form a series of multimers. These multimers appear to contain an even number of subunits of 270K. Two minor components of Mr 140K and 120K were also identified, but these chains appear to result from minor proteolysis. The smallest multimer of vWF contained nearly equimolar amounts of the 270K, 140K, and 120K subunits, while the largest multimers contained less than 20% of the two minor components. Amino acid sequence analysis, amino acid composition, and cleavage by cyanogen bromide indicate that the 270K subunits are identical and each is a single polypeptide chain with an amino-terminal sequence of Ser-Leu-Ser-Cys-Arg-Pro-Pro-Met-Val-Lys and a carboxyl-terminal sequence of Glu-Cys-Lys-Ser-Pro-Arg-Lys-Cys-Ser-Lys. Platelet binding in the presence of ristocetin was 8-fold greater with multimers larger than five (i.e., containing more than 10 subunits of 270K) as compared to multimers less than three (containing less than six subunits of 270K). However, partially reduced vWF (Mr 500K), regardless of whether it was prepared from large or small molecular weight multimers, gave platelet binding similar to that of the smallest multimers. Likewise, partial proteolysis by elastase, thermolysin, trypsin, or chymotrypsin produced small "multimer-like" proteins with platelet binding properties similar to either partially reduced vWF or to the smallest multimers. We conclude that human vWF contains identical 270K subunits assembled into a multivalent structure. Disassembly by either partial reduction or partial proteolysis produces essentially monovalent protein with platelet binding properties similar to that of the smallest multimers. Multivalency is likely the primary factor responsible for the increase in biological activity with multimer size.