一种针对泛素的特异性终点检测法。
A specific endpoint assay for ubiquitin.
作者信息
Rose I A, Warms J V
出版信息
Proc Natl Acad Sci U S A. 1987 Mar;84(6):1477-81. doi: 10.1073/pnas.84.6.1477.
Simple endpoint assays for free ubiquitin (Ub) and for the Ub-activating enzyme are described. The method for measuring Ub makes use of the reaction of iodoacetamide-treated Ub-activating enzyme (E): [3H]ATP + Ub + E----E X [3H]AMP-Ub + PPi and PPi----2Pi (in the presence of pyrophosphatase). The Ub is then measured by determining the acid-insoluble radioactivity. The reaction is accompanied by a slow enzyme-catalyzed hydrolysis of the complex to AMP plus Ub. The presence of ubiquitin-activating enzyme in excess of Ub by approximately equal to 0.1 microM assures that the steady state will be close to the endpoint for total Ub. A preparation of the activating enzyme from human erythrocytes that does not depend on affinity chromatography is described. Several applications of the assay are presented.
本文描述了用于检测游离泛素（Ub）和泛素激活酶的简单终点分析方法。检测Ub的方法利用了碘乙酰胺处理的泛素激活酶（E）的反应：[3H]ATP + Ub + E→E×[3H]AMP-Ub + PPi 以及PPi→2Pi（在焦磷酸酶存在的情况下）。然后通过测定酸不溶性放射性来检测Ub。该反应伴随着复合物缓慢的酶催化水解为AMP和Ub。当泛素激活酶的存在量超过Ub约0.1微摩尔时，可确保稳态接近总Ub的终点。本文还描述了一种不依赖亲和层析从人红细胞中制备激活酶的方法。此外，还介绍了该分析方法的几种应用。
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