A specific endpoint assay for ubiquitin.
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.
D000249:Adenosine Monophosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D006801:Humans; D014316:Tritium; D014452:Ubiquitins / Q000032:analysis* / Q000378:metabolism
D014452:Ubiquitins; D014316:Tritium; D000249:Adenosine Monophosphate; D000255:Adenosine Triphosphate