Williams E B, Krishnaswamy S, Mann K G
Department of Biochemistry, University of Vermont, Burlington 05405.
J Biol Chem. 1989 May 5;264(13):7536-45.
Glutamylglycinylarginyl chloromethyl ketone, tyrosylglycinylarginyl chloromethyl ketone, and phenylalanylprolylarginyl chloromethyl ketone have been labeled at their amino termini using fluorescein, rhodamine-X, lissamine-rhodamine, pyrene, and the 1,5-, 2,5-, and 2,6-dimethylaminonaphthalene-1-sulfonyl moieties. These peptidyl chloromethyl ketones have also been modified by incorporation of biotin and epsilon-amino caproyl biotin. The ability of these various chloromethyl ketones to be incorporated into a collection of zymogen-enzyme pairs has been evaluated using a variety of coagulation and fibrinolytic proteins. All labeled chloromethyl ketones were efficiently incorporated into the proteases tested, with the exception of urokinase which was refractory to inhibition by phenylalanylprolylarginyl chloromethyl ketone derivatives. No modification of any zymogen species was observed even under conditions designed to detect minimal reactivity. When enzymes were modified using chloromethyl ketones labeled with epsilon-amino caproyl biotin, the modified proteins readily reacted with avidin under a variety of different conditions. The observed reactivity with avidin was used in enzyme "blotting" following electrophoretic resolution of polypeptide chains and to remove active enzyme present in enzyme-zymogen mixtures. These reagents have been used to evaluate the potential for active site expression by the single-chain human factor VII molecule. Studies conducted with tissue factor, phospholipids, and calcium using factor X as substrate demonstrate that no activity can be obtained without initial activation of either factor X to factor Xa or factor VII to factor VIIa by an external source. We thus conclude that factor VII is a true zymogen, inert in the blood clotting process prior to its cleavage to factor VIIa.
谷氨酰甘氨酰精氨酰氯甲基酮、酪氨酰甘氨酰精氨酰氯甲基酮和苯丙氨酰脯氨酰精氨酰氯甲基酮已在其氨基末端用荧光素、罗丹明-X、丽丝胺罗丹明、芘以及1,5-、2,5-和2,6-二甲基氨基萘-1-磺酰基部分进行了标记。这些肽基氯甲基酮还通过掺入生物素和ε-氨基己酰生物素进行了修饰。使用多种凝血和纤溶蛋白评估了这些不同氯甲基酮掺入一系列酶原-酶对中的能力。所有标记的氯甲基酮都能有效地掺入所测试的蛋白酶中，但尿激酶除外，它对苯丙氨酰脯氨酰精氨酰氯甲基酮衍生物的抑制作用具有抗性。即使在旨在检测最小反应性的条件下，也未观察到任何酶原种类的修饰。当使用用ε-氨基己酰生物素标记的氯甲基酮修饰酶时，修饰后的蛋白质在各种不同条件下都能与抗生物素蛋白发生反应。在多肽链进行电泳分离后的酶“印迹”中利用观察到的与抗生物素蛋白的反应性，并去除酶-酶原混合物中存在的活性酶。这些试剂已用于评估单链人因子VII分子表达活性位点的潜力。使用因子X作为底物，用组织因子、磷脂和钙进行的研究表明，在没有通过外部来源将因子X初始激活为因子Xa或将因子VII初始激活为因子VIIa的情况下，无法获得活性。因此，我们得出结论，因子VII是一种真正的酶原，在其裂解为因子VIIa之前在血液凝固过程中是无活性的。
