title: A comparative study of the biochemical properties of human and mouse recombinant O6-methylguanine-DNA methyltransferases.
abstract: The O6-methylguanine-DNA methyltransferase (MGMT) repairs mutagenic and carcinogenic O6-alkylguanine in DNA by accepting stoichiometrically the alkyl group from the base. Although the mouse MGMT is larger than the human protein because of an additional tetrapeptide sequence, these proteins are 70% homologous. Recombinant MGMTs of the human, the mouse and a mouse mutant with the tetrapeptide deleted were purified to homogeneity from Escherichia coli. The N-terminal amino acid sequences of these proteins are identical to those predicted from the nucleotide sequences, and their molecular masses determined by SDS-PAGE agreed with the predicted values. However, the observed isoelectric points of 9.3, 9.2 and 9.3, for the human, mouse and mutant mouse proteins respectively were significantly different from the values, 8.09, 7.47 and 7.49 calculated from the amino acid composition. The extinction coefficients E280 nm1% of human, mouse and mutant mouse protein were calculated from amino acid composition to be 18.2, 11.1 and 11.3 respectively. These values agree fairly well with calculated values. Human and wild-type mouse MGMTs react with the alkylated base in a synthetic DNA substrate poly(dC, dG, m6dG) with comparable second-order rate constants of 2.2 x 10(8) and 3.7 x 10(8) l/M/min at 37 degrees C respectively and were inactivated by O6-benzylguanine at similar rates. The initial reaction rate (Kin) and rate of inactivation (kinact) constants for reaction with the base were calculated to be 1.8 x 10(-4) M and 1.4 x 10(-3)/s for the human protein, 2.3 x 10(-4) M and 1.1 x 10(-3)/s for the wild-type mouse protein, and 2.1 x 10(-4) M and 1.4 x 10(-3)/s for the mutant mouse protein respectively. The MGMTs were inactivated to the extent of 55-65% after heating at 50 degrees C in 20 mM Tris-HCl, pH 8.0, 1 mM EDTA, 1 mM DTT and 10% glycerol. However, in the presence of DNA (200 micrograms/ml), only 25-35% of the protein was inactivated. Both DNA and RNA inhibited all three enzymes in a concentration-dependent fashion, although DNA was a better inhibitor than RNA. High salt (0.2 M NaCl) inhibited human MGMT by 80%, while the wild-type and the mutant mouse MGMTs were inhibited by 55%. The human protein had higher affinity for binding to duplex DNAs than the mouse proteins. Immunoprecipitation (69%) and affinity constant (19.4 nM) of human MGMT with a human-specific monoclonal antibody 4.A1 significantly discriminated the human protein from either of the mouse proteins.
authors.affiliation: Sealy Center for Molecular Sciences, University of Texas Medical Branch, Galveston 77555.
authors.forename: R
authors.initials: R
authors.lastname: Roy
authors.forename: S
authors.initials: S
authors.lastname: Shiota
authors.forename: S J
authors.initials: SJ
authors.lastname: Kennel
authors.forename: R
authors.initials: R
authors.lastname: Raha
authors.forename: M
authors.initials: M
authors.lastname: von Wronski
authors.forename: T P
authors.initials: TP
authors.lastname: Brent
authors.forename: S
authors.initials: S
authors.lastname: Mitra
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D002417:Cattle; D002482:Cellulose / Q000031:analogs & derivatives / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D018604:Epitope Mapping; D006801:Humans; D007526:Isoelectric Point; D007700:Kinetics; D008780:Methyltransferases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000502:physiology*; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009154:Mutation; D019853:O(6)-Methylguanine-DNA Methyltransferase; D011233:Precipitin Tests; D012313:RNA / Q000378:metabolism; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; C033190:DNA-cellulose; D011994:Recombinant Proteins; D012313:RNA; D002482:Cellulose; D004247:DNA; D008780:Methyltransferases; D019853:O(6)-Methylguanine-DNA Methyltransferase