Partial purification and characterization of 3-methyladenine-DNA glycosylase from human placenta.
A DNA glycosylase was isolated and purified over 1000-fold from human placentas by means of diethylamino-ethylcellulose and double- and single-stranded DNA-Sepharose affinity chromatography. The procedure was rapid and yielded greater than 15% of the initial enzyme activity. High-pressure liquid chromatographs of reaction products showed that 3-methyladenine was the predominant substrate in methylated native DNA. 7-Methylguanine and 3-methylguanine were also released by the partially purified enzyme, albeit at low rates; release was more evident when the substrate was methylated double-stranded poly(dG-dC). The enzyme preparation was essentially free of nuclease activity, retaining less than 0.001% of the initial cellular concentration of Mg2+-requiring apurinic endonuclease activity. The glycosylase had a broad pH optimum between 7.2 and 7.7; it did not require metal ions but was stimulated by Na+ or K+ at 50 mM or by Mg2+ at 1 mM. Higher concentrations of these ions were inhibitory. Activity was unaffected by beta-mercaptoethanol or dithiothreitol, but 1 mM N-ethylmaleimide or p-(hydroxymercuri)benzoate as well as 1 mM spermine or 10 mM sperimidine totally inhibited the enzyme. The apparent molecular weight of the glycosylase, determined by gel filtration, was 25000, and the apparent Km for 3-methyladenine in native methylated DNA was 3 x 10(-8) M. The enzyme required double-stranded methylated DNA as a substrate and showed very low activity with denatured methylated DNA. It appeared that single-stranded regions in DNA inhibited 3-methyladenine-DNA glycosylase activity, but up to 1 mM concentrations of free methylated bases did not.
D000225:Adenine / Q000031:analogs & derivatives / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D045647:DNA Glycosylases; D005260:Female; D006147:Guanine / Q000031:analogs & derivatives / Q000378:metabolism; D006801:Humans; D009699:N-Glycosyl Hydrolases / Q000302:isolation & purification* / Q000378:metabolism; D010920:Placenta / Q000201:enzymology*; D011247:Pregnancy; D013379:Substrate Specificity
C025946:3-methyladenine; D006147:Guanine; C008450:7-methylguanine; C021565:3-methyladenine-DNA glycosylase; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; D000225:Adenine