Roy R, Shiota S, Kennel S J, Raha R, von Wronski M, Brent T P, Mitra S
Sealy Center for Molecular Sciences, University of Texas Medical Branch, Galveston 77555.
Carcinogenesis. 1995 Feb;16(2):405-11. doi: 10.1093/carcin/16.2.405.
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.
O6-甲基鸟嘌呤-DNA甲基转移酶（MGMT）通过化学计量地从碱基接受烷基来修复DNA中具有致突变性和致癌性的O6-烷基鸟嘌呤。尽管由于额外的四肽序列，小鼠MGMT比人类蛋白更大，但这些蛋白具有70%的同源性。人、小鼠以及缺失四肽的小鼠突变体的重组MGMT从大肠杆菌中纯化至同质。这些蛋白的N端氨基酸序列与从核苷酸序列预测的序列相同，通过SDS-PAGE测定的它们的分子量与预测值相符。然而，人、小鼠和突变小鼠蛋白观察到的等电点分别为9.3、9.2和9.3，与根据氨基酸组成计算出的8.09、7.47和7.49的值显著不同。根据氨基酸组成计算出人、小鼠和突变小鼠蛋白在280nm波长下的消光系数E280nm1%分别为18.2、11.1和11.3。这些值与计算值相当吻合。人及野生型小鼠MGMT与合成DNA底物聚（dC，dG，m6dG）中的烷基化碱基反应，在37℃时二级速率常数分别为2.2×10⁸和3.7×10⁸ l/M/min，且被O6-苄基鸟嘌呤以相似速率灭活。人蛋白与碱基反应的初始反应速率（Kin）和失活速率（kinact）常数经计算分别为1.8×10⁻⁴ M和1.4×10⁻³/s，野生型小鼠蛋白为2.3×10⁻⁴ M和1.1×10⁻³/s，突变小鼠蛋白为2.1×10⁻⁴ M和1.4×10⁻³/s。在20mM Tris-HCl，pH 8.0，1mM EDTA，1mM DTT和10%甘油中5℃加热后，MGMT失活程度达55 - 65%。然而，在存在DNA（200μg/ml）时，只有25 - 35%的蛋白失活。DNA和RNA均以浓度依赖方式抑制这三种酶，尽管DNA是比RNA更好的抑制剂。高盐（0.2M NaCl）抑制人MGMT达80%，而野生型和突变小鼠MGMT被抑制55%。人蛋白与双链DNA结合的亲和力高于小鼠蛋白。人MGMT与人特异性单克隆抗体4.A1的免疫沉淀率（69%）和亲和常数（19.4nM）显著区分了人蛋白与任一小鼠蛋白。
