Bittorf S V, Williams E C, Mosher D F
Department of Medicine, University of Wisconsin, Madison 53706.
J Biol Chem. 1993 Nov 25;268(33):24838-46.
Vitronectin circulates in blood as a 70-kDa monomer that interacts with complexes generated in the terminal steps of the coagulation and complement cascades. Vitronectin complexed to thrombin-antithrombin or C5b-9 is conformationally altered as evidenced by enhanced reactivity with monoclonal antibody 8E6 and binding to heparin; these same alterations also occur when vitronectin is treated with urea (Tomasini, B. R., and Mosher, D.F. (1988) Blood 72, 903-912; Høgåsenk, K., Molnes, T.E., and Harboe, M. (1992) J. Biol. Chem. 267, 23076-23082). We have modified the purifications of native and urea-treated vitronectin to better control conformational state and characterized the alterations induced by urea. After treatment with N-ethylmaleimide to prevent formation of disulfide-linked multimers, purification in 8 M urea, and dialysis against physiological saline, vitronectin was largely oligomeric (approximately 800 kDa) as assessed by gel filtration and polyacrylamide gel electrophoresis in the absence of sodium dodecyl sulfate. Oligomeric urea-treated vitronectin reacted more strongly with the 8E6 antibody, bound biotinylated heparin more strongly, and neutralized the anticoagulant activity of heparin better than monomeric altered vitronectin or native vitronectin. After incubation with urea at 25 degrees C, native vitronectin, treated during purification with dithionitrobenzoic acid to force free sulfhydryls to intramolecular disulfides, exhibited increased reactivity with antibody 8E6, increased binding to heparin, and oligomerization. In addition, incubation in urea caused rearrangement of disulfides as assessed by loss of the light chain of two-chain vitronectin. The transition for these effects occurred between 2 and 4 M urea. Thus, an irreversible conformational alteration occurs upon treatment of vitronectin with urea, resulting in oligomers that bind avidly to heparin.
玻连蛋白以70 kDa的单体形式在血液中循环，它与凝血和补体级联反应终末步骤中产生的复合物相互作用。与凝血酶 - 抗凝血酶或C5b - 9复合的玻连蛋白在构象上发生了改变，这可通过与单克隆抗体8E6反应性增强以及与肝素结合得到证明；当玻连蛋白用尿素处理时也会出现同样的改变（托马西尼，B.R.，和莫舍，D.F.（1988年）《血液》72，903 - 912；霍格森，K.，莫尔内斯，T.E.，和哈博，M.（1992年）《生物化学杂志》267，23076 - 23082）。我们改进了天然和尿素处理的玻连蛋白的纯化方法，以更好地控制构象状态，并对尿素诱导的改变进行了表征。在用N - 乙基马来酰亚胺处理以防止形成二硫键连接的多聚体后，在8 M尿素中纯化，并对生理盐水进行透析，通过凝胶过滤和在无十二烷基硫酸钠情况下的聚丙烯酰胺凝胶电泳评估，玻连蛋白在很大程度上是寡聚体（约800 kDa）。与单体形式改变的玻连蛋白或天然玻连蛋白相比，寡聚体形式的尿素处理玻连蛋白与8E6抗体反应更强，与生物素化肝素结合更强，并且能更好地中和肝素的抗凝血活性。在25℃用尿素孵育后，在纯化过程中用二硫代硝基苯甲酸处理以迫使游离巯基形成分子内二硫键的天然玻连蛋白，与抗体8E6的反应性增加，与肝素的结合增加，并且发生了寡聚化。此外，通过双链玻连蛋白轻链的丢失评估，在尿素中孵育导致了二硫键的重排。这些效应的转变发生在2至4 M尿素之间。因此，用尿素处理玻连蛋白会发生不可逆的构象改变，产生与肝素 avidly 结合的寡聚体。 （注：avidly这个词原文可能有误，推测可能是avidly，意为“贪婪地、热切地”，这里暂且保留原文形式，若有误请确认原文后修改翻译）
