Identification of the EcoKI and EcoR124I Type I restriction--modification enzyme subunits by non-equilibrium pH gradient two-dimensional gel electrophoresis.
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.
Nguyen
L D
LD
Institute of Microbiology, Academy of Sciences of the Czech Republic, 142 20 Prague, Czechia.
Cajthamlová
K
K
Nguyen
H T
HT
Weiser
J
J
Holubová
I
I
Weiserová
M
M
D015153:Blotting, Western; D004262:DNA Restriction Enzymes / Q000737:chemistry; D015280:DNA Restriction-Modification Enzymes / Q000737:chemistry*; D015253:Deoxyribonucleases, Type I Site-Specific / Q000737:chemistry; D015180:Electrophoresis, Gel, Two-Dimensional; D004926:Escherichia coli / Q000201:enzymology*; D007526:Isoelectric Point; D008970:Molecular Weight; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific) / Q000737:chemistry
D015280:DNA Restriction-Modification Enzymes; C093244:DNA modification methylase EcoKI; C072994:EcoR124 modification methylase type I; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific); D004262:DNA Restriction Enzymes; C089629:endodeoxyribonuclease EcoKI; C048242:endodeoxyribonuclease EcoR124I; D015253:Deoxyribonucleases, Type I Site-Specific