Induction and purificattion of O6-methylguanine-DNA-methyltransferase from rat liver

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Abstract
O6-Methylguanine-DNA-methyltransferase (OMMT) is a DNA repair protein that plays an important role in chemotherapy, mutagenesis and carcinogenesis. The sp. act. of OMMT in rat liver can be induced by ∼12-to 20-fold by treatment of the rats with ionizing radiation. The effects of dose and time were investigated in this study. We have found that OMMT sp. act. can be increased, although to a lower extent, in kidney, spleen and brain in addition to liver. However, the sp. act. of OMMT in lung was reduced by irradiation. OMMT has been purified from the livers of irradiated rats by solubilization in high-salt-containing buffer, ammomum sulfate precipitation and a series of column chromatographic steps, including phenyl-Sepharose, heparin-agarose, double-stranded DNA-cellulose and FPLC. A 3000-fold enrichment of OMMT was achieved from the induced liver preparations. However, with regard to the sp. act. of this protein in normal rat liver, the fold purification was ∼35 000. After methylation, OMMT during the course of its action exhibited a mol. wt of 28 kd under SDS-PAGE conditions..