Identification of the EcoKI and EcoR124I Type I restriction--modification enzyme subunits by non-equilibrium pH gradient two-dimensional gel electrophoresis
Language English Country United States Media print
Document type Journal Article, Research Support, Non-U.S. Gov't
PubMed
12630312
DOI
10.1007/bf02818664
Knihovny.cz E-resources
- MeSH
- Electrophoresis, Gel, Two-Dimensional MeSH
- DNA Restriction-Modification Enzymes chemistry MeSH
- Escherichia coli enzymology MeSH
- Isoelectric Point MeSH
- Site-Specific DNA-Methyltransferase (Adenine-Specific) chemistry MeSH
- Molecular Weight MeSH
- Deoxyribonucleases, Type I Site-Specific chemistry MeSH
- DNA Restriction Enzymes chemistry MeSH
- Blotting, Western MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- DNA modification methylase EcoKI MeSH Browser
- DNA Restriction-Modification Enzymes MeSH
- EcoR124 modification methylase type I MeSH Browser
- endodeoxyribonuclease EcoKI MeSH Browser
- endodeoxyribonuclease EcoR124I MeSH Browser
- Site-Specific DNA-Methyltransferase (Adenine-Specific) MeSH
- Deoxyribonucleases, Type I Site-Specific MeSH
- DNA Restriction Enzymes MeSH
Effectively optimized and reproducible procedure for monitoring the composition of type I restriction-modification endonucleases EcoKI and EcoR124I by non-equilibrium pH gradient two-dimensional (2-D) gel electrophoresis is described. Three subunits of the enzyme complex, which widely differ from one another in their isoelectric points and molar mass, were identified in crude cell extracts of E. coli. For the first time all three subunits of both EcoKI and EcoR124I were detected as distinct spots on a single 2-D gel. A sensitive immunoblotting procedure was suggested suitable for routine use in determining the identity of individual subunits. Potential application of this method for detailed studies of regulation of the function and stoichiometry of the enzyme complexes is discussed.
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