Quantitation of S-methylcysteine formed in O6-methylguanine-DNA:methyltransferase.
The O6-methylguanine-DNA:methyltransferase is known to transfer the methyl group from O6-methylguanine (O6-mG) in DNA to the cysteinyl residue of the methyl acceptor protein which is the methyltransferase itself. We developed a novel method to estimate the amount of S-methylcysteine formed in the acceptor protein, utilizing O6-[methyl-3H]methylguanine-containing DNA as the methyl donor. Following hydrolysis of the methyl-incorporated macromolecules in formic acid--HCl, S-[methyl-3H]methylcysteine is chromatographically isolated on a small column of Dowex-50(H+). Utilizing this method, we measured O6-mG DNA:methyltransferase activity in rat liver during neonatal development; the activity increases from 0.013 pmol methyl transferred/mg protein at 2 days postnatally to 0.06 pmol/mg at 8 weeks, the latter being equal to the adult liver activity.
D000367:Age Factors; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D000831:Animals, Newborn; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003545:Cysteine / Q000031:analogs & derivatives* / Q000032:analysis; D004247:DNA / Q000378:metabolism*; D004260:DNA Repair; D008099:Liver / Q000201:enzymology*; D008780:Methyltransferases / Q000378:metabolism*; D019853:O(6)-Methylguanine-DNA Methyltransferase; D011496:Protein Methyltransferases / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains
D000596:Amino Acids; D004247:DNA; C008425:S-methylcysteine; D008780:Methyltransferases; D011496:Protein Methyltransferases; D019853:O(6)-Methylguanine-DNA Methyltransferase; D003545:Cysteine