A COMPARATIVE-STUDY OF THE BIOCHEMICAL-PROPERTIES OF HUMAN AND MOUSE RECOMBINANT O-6-METHYLGUANINE-DNA METHYLTRANSFERASES

The O-6-methylguanine-DNA methyltransferase (MGMT) repairs mutagenic a nd carcinogenic O-6-alkylguanine in DNA by accepting stoichiometricall y the alkyl group from the base. Although the mouse MGMT is larger tha n the human protein because of an additional tetrapeptide sequence, th ese proteins are 70 % homologous. Recombinant MGMTs of the human, the mouse and a mouse mutant with the tetrapeptide deleted were purified t o homogeneity from Escherichia coli. The N-terminal amino acid sequenc es of these proteins are identical to those predicted from the nucleot ide sequences, and their molecular masses determined by SDS-PAGE agree d 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 re spectively were significantly different from the values, 8.09, 7.47 an d 7.49 calculated from the amino acid composition. The extinction coef ficients E(1%)(280 nm) of human, mouse and mutant mouse protein were c alculated from amino acid composition to be 18.2, 11.1 and 11.3 respec tively. These values agree fairly well with calculated values. Human a nd wild-type mouse MGMTs react with the alkylated base in a synthetic DNA substrate poly(dC, dG, m(6)dG) with comparable second-order rate c onstants of 2.2x10(8) and 3.7x10(8) 1/M/min at 37 degrees C respective ly and were inactivated by O-6-benzylguanine at similar rates. The ini tial reaction rate (K-in) and rate of inactivation (k(inact)) constant s for reaction with the base were calculated to be 1.8X10(-4) M and 1. 4X10(-3)/s for the human protein, 2.3x10(-4) M and 1.1X10(-3)/s for th e wild-type mouse protein, and 2.1X10(-4) M and 1.4x10(-3)/s for the m utant mouse protein respectively. The MGMTs were inactivated to the ex tent 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 mu g/ml), only 25-35% of the protein was inactivated. Both D NA and RNA inhibited all three enzymes in a concentration-dependent fa shion, 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 affini ty for binding to duplex DNAs than the mouse proteins. Immunoprecipita tion (69%) and affinity constant (19.4 nM) of human MGMT with a human- specific monoclonal antibody 4.A1 significantly discriminated the huma n protein from either of the mouse proteins.