title: Purification to apparent homogeneity and partial amino acid sequence of rat liver O6-alkylguanine-DNA-alkyltransferase.
abstract: O6-alkylguanine-DNA-alkyltransferase (ATase) activity was increased in rat liver from 80 to 320 fmoles/mg total protein 48 h after administration of 2-acetylaminofluorene at 60 mg/kg body weight. This tissue was used as a source of ATase which was purified by ammonium sulphate precipitation and DNA-cellulose, molecular exclusion and ion exchange chromatography (IEC). IEC purified material showed a major 24 kDa band after polyacrylamide gel electrophoresis (PAGE) with silver staining. Fluorography of purified ATase following incubation with [3H]-methylated substrate DNA and PAGE showed a single band at 24 kDa suggesting that, as with bacterial ATases, the protein itself accepts the alkyl group from O6-alkylguanine in substrate DNA during the repair reaction. Further purification of the protein using reverse phase HPLC resulted in a single peak representing approximately 125,000 fold purification. This was subjected to amino-terminal sequencing and it was found that the protein was blocked at the amino-terminal end: it was cleaved using trypsin or cyanogen bromide and the amino acid sequence of several reverse phase HPLC purified fragments was determined.
authors.affiliation: Department of Carcinogenesis, Paterson Institute for Cancer Research, Christie Hospital, Manchester, UK.
authors.forename: M C
authors.initials: MC
authors.lastname: Wilkinson
authors.forename: D P
authors.initials: DP
authors.lastname: Cooper
authors.forename: C
authors.initials: C
authors.lastname: Southan
authors.forename: P M
authors.initials: PM
authors.lastname: Potter
authors.forename: G P
authors.initials: GP
authors.lastname: Margison
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D008099:Liver / Q000201:enzymology; D008780:Methyltransferases / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D019853:O(6)-Methylguanine-DNA Methyltransferase; D010446:Peptide Fragments / Q000032:analysis; D051381:Rats
chemical_list: D010446:Peptide Fragments; D008780:Methyltransferases; D019853:O(6)-Methylguanine-DNA Methyltransferase