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PMID: 3031643 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

A specific endpoint assay for ubiquitin.

Proceedings of the National Academy of Sciences of the United States of America ·Vol. 84 ·No. 6 ·1987-03-00 ·Pages 1477-81

Rose IA, Warms JV

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.

MeSH Terms
Adenosine Monophosphate/metabolism Adenosine Triphosphate/metabolism Humans Tritium Ubiquitins/analysis,metabolism
Chemicals
Ubiquitins Tritium Adenosine Monophosphate Adenosine Triphosphate
Authors & Affiliations
2 authors, click to expand affiliations / ORCID
Rose I A
Warms J V
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24 references, click to expand
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Article Info
Journal
Proceedings of the National Academy of Sciences of the United States of America
Abbr.
Proc Natl Acad Sci U S A
ISSN
0027-8424
DOI
10.1073/pnas.84.6.1477
Published
1987-03-00
Pages
1477-81
Language
English
Region
United States
NLM ID
7505876
PMCID
PMC304457
Subset
IM
Grants
NCI NIH HHS · CA-06927 · United States
NCRR NIH HHS · RR-05539 · United States
External Links
PubMed source DOI: 10.1073/pnas.84.6.1477 PMC Full Text View journal details
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