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Literature DB >> 3031643

A specific endpoint assay for ubiquitin.

Abstract

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.

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Year: 1987 PMID： 3031643 PMCID： PMC304457 DOI： 10.1073/pnas.84.6.1477

Source DB: PubMed Journal: Proc Natl Acad Sci U S A ISSN： 0027-8424 Impact factor: 11.205

24 in total

A specific endpoint assay for ubiquitin.

1. Isolation and characterization of protein A24, a "histone-like" non-histone chromosomal protein.

2. Multiple forms of ubiquitin-protein ligase. Binding of activated ubiquitin to protein substrates.

3. Stimulation of ATP-dependent proteolysis requires ubiquitin with the COOH-terminal sequence Arg-Gly-Gly.

4. Metabolism of ubiquitinated histones.

5. Histone 2B can be modified by the attachment of ubiquitin.

6. Characterization of the heat-stable polypeptide of the ATP-dependent proteolytic system from reticulocytes.

7. Chromatin conjugate protein A24 is cleaved and ubiquitin is lost during chicken erythropoiesis.

8. Selective arrangement of ubiquitinated and D1 protein-containing nucleosomes within the Drosophila genome.

9. "Covalent affinity" purification of ubiquitin-activating enzyme.

10. Activation of the heat-stable polypeptide of the ATP-dependent proteolytic system.

1. Ubiquitin-aldehyde: a general inhibitor of ubiquitin-recycling processes.

2. Ubiquitin fusion augments the yield of cloned gene products in Escherichia coli.

3. Inhibition of proteolysis and cell cycle progression in a multiubiquitination-deficient yeast mutant.

4. Ubiquitin depletion as a key mediator of toxicity by translational inhibitors.

5. A spectrophotometric assay for conjugation of ubiquitin and ubiquitin-like proteins.

6. Ubiquitin charging of human class III ubiquitin-conjugating enzymes triggers their nuclear import.