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首页> 外文期刊>Biochemistry >Phosphorothioate Oligonucleotides Inhibit the Intrinsic Tenase Complex by an Allosteric Mechanism
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Phosphorothioate Oligonucleotides Inhibit the Intrinsic Tenase Complex by an Allosteric Mechanism

机译：硫代磷酸酯寡核苷酸通过变构机制抑制内在肌腱酶复合物。

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Phosphorothioate oligonucleotides (PS ODNs) prolong the activated partial thromboplastin time in human plasma by inhibition of intrinsic tenase (factor IXa—factor Villa) activity. This inhibition was characterized using ISIS 2302, a 20-mer antisense PS ODN. ISIS 2302 demonstrated hyperbolic, mixed-type inhibition of factor X activation by the intrinsic tenase complex. The decrease in Vmax(app) was analyzed by examining complex assembly, cofactor stability, and protease catalysis. ISIS 2302 did not inhibit factor X activation by the factor IXa—phospholipid complex, or significantly affect factor VIII— phospholipid affinity. Inhibitory concentrations of ISIS 2302 modestly decreased the affinity of factor IXa—factor Villa binding in the presence of phospholipid (KD 11.5 vs 4.8 nM). This effect was insufficient to explain the reduction in Vmax(app). ISIS 2302 did not affect the in vitro half-life of factor Villa, suggesting it did not destabilize cofactor activity. In the presence of 30% ethylene glycol, the level of factor X activation by the factor IXa—phospholipid complex increased 3-fold, and the level of chromogenic substrate cleavage by factor IXa increased more than 50-fold. ISIS 2302 demonstrated partial inhibition of factor X activation by the factor IXa—phospholipid complex, and chromogenic substrate cleavage by factor IXa, only in the presence of ethylene glycol. Like the intact enzyme complex, ISIS 2302 demonstrated hyperbolic, mixed-type inhibition of chromogenic substrate cleavage by factor IXa (K1 88 nM). Equilibrium binding studies with fluorescein-labeled ISIS 2302 demonstrated a similar affinity (KD 92 nM) for the PS ODN—factor IX interaction. These results suggest that PS ODNs bind to an exosite on factor IXa, modulating catalytic activity of the intrinsic tenase complex.
机译：硫代磷酸酯寡核苷酸（PS ODN）通过抑制固有肌腱酶（因子IXa-因子Villa）活性来延长人血浆中活化的部分凝血活酶时间。使用20聚体反义PS ODN ISIS 2302表征了这种抑制作用。 ISIS 2302通过固有的肌腱酶复合物证明了对因子X激活的双曲线混合型抑制。通过检查复杂的装配，辅因子稳定性和蛋白酶催化分析了Vmax（app）的降低。 ISIS 2302不会通过因子IXa-磷脂复合物抑制因子X的激活，也不会显着影响因子VIII-磷脂的亲和力。在磷脂存在下，ISIS 2302的抑制浓度会适度降低因子IXa-因子Villa结合的亲和力（KD 11.5对4.8 nM）。这种效果不足以解释Vmax（app）的降低。 ISIS 2302不会影响因子Villa的体外半衰期，表明它不会破坏辅助因子的活性。在存在30％乙二醇的情况下，由因子IXa-磷脂复合物激活的因子X的水平增加了3倍，而由因子IXa裂解的生色底物的水平增加了50倍以上。 ISIS 2302仅在乙二醇的存在下证明了因子IXa-磷脂复合物对因子X活化的部分抑制，以及因子IXa的生色底物裂解。像完整的酶复合物一样，ISIS 2302对因子IXa（K1 88 nM）的生色底物裂解表现出了双曲线的混合型抑制作用。用荧光素标记的ISIS 2302进行的平衡结合研究表明，对PS ODN-IX因子的相互作用具有相似的亲和力（KD 92 nM）。这些结果表明，PS ODNs与因子IXa上的外位结合，从而调节内在酶复合物的催化活性。

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