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.