Hypoxanthine-DNA glycosylase from Escherichia coli. Partial purification and properties.
Hypoxanthine-DNA glycosylase from Escherichia coli was partially purified by ammonium sulfate fractionation and by chromatography on Sephacryl S-200, DEAE-cellulose, and phosphocellulose P-11 columns. Analysis of the enzymatic reaction products was carried out on a minicolumn of DEAE-cellulose and/or by paper chromatography, by following the release of the free base [3H]hypoxanthine from [3H]dIMP-containing phi X174 DNA. In native conditions, the enzyme has a molecular mass of 60 +/- 4 kDa, as determined by gel filtration on Sephadex G-150 and Sephacryl S-200 columns. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed a major polypeptide band of an apparent molecular mass of 56 kDa, and glycerol gradient centrifugation indicated a sedimentation coefficient of 4.0 S. Hypoxanthine-DNA glycosylase from E. coli has an obligatory requirement for Mg2+ and is totally inhibited in the presence of EDTA. Co2+ can only partially replace Mg2+. The enzyme is inhibited by hypoxanthine which at 4 mM causes 85% inhibition. The optimal pH range of the enzymatic activity is 5.5-7.8, and the apparent Km value is 2.5 x 10(-7) M.
D000645:Ammonium Sulfate; D010584:Bacteriophage phi X 174; D002413:Cations, Divalent; D002499:Centrifugation, Density Gradient; D002845:Chromatography; D002854:Chromatography, Paper; D004279:DNA, Viral / Q000378:metabolism; D004492:Edetic Acid / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology*; D005590:Fractional Precipitation; D006026:Glycoside Hydrolases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D019271:Hypoxanthine; D007042:Hypoxanthines / Q000378:metabolism / Q000494:pharmacology; D007291:Inosine Monophosphate / Q000031:analogs & derivatives / Q000378:metabolism; D008274:Magnesium / Q000494:pharmacology; D008970:Molecular Weight; D013307:Streptomycin; D013379:Substrate Specificity; D013696:Temperature
D002413:Cations, Divalent; D004279:DNA, Viral; D007042:Hypoxanthines; D007291:Inosine Monophosphate; D019271:Hypoxanthine; C017193:deoxyinosine monophosphate; D004492:Edetic Acid; D006026:Glycoside Hydrolases; C035336:DNA-deoxyinosine glycosidase; D008274:Magnesium; D000645:Ammonium Sulfate; D013307:Streptomycin