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首页> 外文期刊>Biochemistry >Functional Properties of Recombinant Factor V Mutated in a Potential Calcium-Binding Site
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Functional Properties of Recombinant Factor V Mutated in a Potential Calcium-Binding Site

机译：潜在的钙结合位点中突变的重组因子V的功能特性。

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Activated coagulation factor V (FVa) is a cofactor of activated factor X (FXa) in prothrombin activation.FVa is composed of a light chain (LC) and a heavy chain (HC) that are noncovalently associated in a calcium-dependent manner.We constructed a recombinant FV Aspl 1 lAsn/Aspl 12Asn mutant (rFV-NN) to abolish calcium binding to a potential calcium-binding site in FVa in order to study the specific role of these residues in the expression of FVa activity.Whereas thrombin-activated recombinant FV wild type (rFV-wt) presented with stable FVa activity,incubation of rFV-NN with thrombin resulted in a temporary increase in FVa activity,which was rapidly lost upon prolonged incubation.Loss of FVa activity was most likely due to dissociation of HC and LC since,upon chromatography of rFVa-NN on a SP-Sepharose column,the HC did not bind significantly to the resin whereas the LC bound and could be eluted at high ionic strength.In contrast,rFVa-wt adhered to the column,and both the HC and LC coeluted at high ionic strength.In the presence of phospholipid vesicles,the loss of rFVa-NN activity was partially prevented by FXa,active site inhibited FXa,and prothombin in a dose-dependent manner.We conclude that the introduced amino acid substitutions result in a loss of the high-affinity (calcium-dependent) interaction of the HC and LC of FVa.We propose that the introduced substitutions disrupt the calcium-binding site in FV,thereby yielding a FV molecule that rapidly loses activity following thrombin-catalyzed activation most likely via dissociation of the HC and LC.
机译：活化凝血因子V（FVa）是凝血酶原活化中活化因子X（FXa）的辅因子.FVa由轻链（LC）和重链（HC）组成，它们以钙依赖性方式非共价结合。为了研究这些残基在FVa活性表达中的特定作用，构建了一个重组FV Aspl 1 lAsn / Aspl 12Asn突变体（rFV-NN）以消除钙与FVa中潜在的钙结合位点的结合。重组FV野生型（rFV-wt）具有稳定的FVa活性，rFV-NN与凝血酶的孵育导致FVa活性的暂时增加，长时间孵育会迅速丧失.FVa活性的损失很可能是由于HC和LC，因为在SP-Sepharose色谱柱上进行rFVa-NN色谱分析时，HC未明显结合到树脂上，而LC却被结合并可以高离子强度洗脱。相反，rFVa-wt附着在色谱柱上，HC和LC线圈在存在磷脂囊泡的情况下，FXa，活性位点抑制的FXa和凝血酶原均以剂量依赖的方式部分阻止了rFVa-NN活性的丧失。我们得出结论，引入的氨基酸取代导致FVa的HC和LC的高亲和力（依赖钙的）相互作用的丧失。我们建议引入的取代破坏FV中的钙结合位点，从而产生FV分子，该分子在凝血酶催化的激活后迅速丧失活性最有可能是通过HC和LC的解离。

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