Comparison of the inactivation of mammalian and bacterial O6-alkylguanine-DNA alkyltransferases by O6-benzylguanine and O6-methylguanine.
Comparison of the inactivation of mammalian and bacterial O6-alkylguanine-DNA alkyltransferases by O6-benzylguanine and O6-methylguanine.
复制标题
O6-苄基鸟嘌呤和 O6-甲基鸟嘌呤对哺乳动物和细菌 O6-烷基鸟嘌呤-DNA 烷基转移酶的灭活作用的比较。
DOI：
10.1093/carcin/12.12.2305
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发表时间：
1991
期刊：
影响因子：
4.7
通讯作者：
Gerson,SL
中科院分区：
文献类型：
--
作者：
Dolan,ME;Pegg,AE;Dumenco,LL;Moschel,RC;Gerson,SL
比较了o6 -甲基鸟嘌呤和do6 -苄基鸟嘌呤对人和大肠杆菌6-烷基鸟嘌呤- dna烷基转移酶的失活作用。HT29细胞提取物orE.coliAda蛋白在200 μ mo6 -甲基鸟嘌呤中孵育1 h，烷基转移酶活性分别降至对照水平的44%和39%。然而，在相同的条件下，so6 -苄基鸟嘌呤完全降低了人细胞提取物的烷基转移酶活性，但对Ada蛋白几乎没有影响。HT29细胞烷基转移酶与th6 -苄基[3H]鸟嘌呤的孵育导致[3H]鸟嘌呤的产生具有时间依赖性。在Ada蛋白存在的情况下，没有观察到类似的[3H]鸟嘌呤的产生。在转染细菌ada基因（CHO-ada）或人烷基转移酶cDNA （CHO- mgmt）的CHO细胞中，500 μ mo6 -甲基鸟嘌呤对这两种烷基转移酶的抑制作用均达到85%。2 μ mo6 -苄基鸟嘌呤可抑制CHO-MGMT细胞中人烷基转移酶的表达，抑制率为bb0 99%，但在25 μM浓度下作用24 h对CHO-ada细胞中细菌烷基转移酶的表达无抑制作用。在肝脏中表达ada的转基因小鼠中也观察到这种选择性抑制作用，其中o6 -苄基鸟嘌呤导致肝脏总烷基转移酶活性仅下降40%，而非转基因小鼠则降低95%，这与仅抑制哺乳动物烷基转移酶和维持细菌烷基转移酶活性的结果一致。因此，虽然高浓度的eo6 -甲基鸟嘌呤使细菌和哺乳动物的烷基转移酶失活，但o6 -苄基鸟嘌呤仅作为哺乳动物蛋白的底物，可能由于位阻，无法抑制Ada蛋白。
The inactivation of human and Escherichia coliO6-alkylguanine-DNA alkyltransferase byO6-methylguanine andO6-benzylguanine was compared. When HT29 cell extracts orE.coliAda protein were incubated in the presence of 200 μMO6-methylguanine for 1 h, alkyltransferase activity was reduced to 44 and 39% of control levels respectively. However, under the same conditionsO6-benzylguanine completely depleted alkyltransferase activity in the extract from human cells but had virtually no effect on the Ada protein. Incubation of the HT29 cell alkyltransferase withO6-benzyl[3H]guanine resulted in a time-dependent production of [3H]guanine. No similar production of [3H]guanine was observed in the presence of the Ada protein. In CHO cells transfected with the bacterial ada gene (CHO-ada) or the human alkyltransferase cDNA (CHO-MGMT), treatment with 500 μMO6-methylguanine inhibited both alkyltransferases by >85%. In contrast, 2 μMO6-benzylguanine inhibited human alkyltransferase expressed in CHO-MGMT cells by >99% though concentrations as high as 25 μM for 24 h had no inhibitory effects on the bacterial akyltransferase expressed in CHO-ada cells. This selective inhibition was also observedin vivoin transgenic mice expressing ada in the liver whereO6-benzylguanine caused a decrease of only 40% in total hepatic alkyltransferase activity compared to 95% in non-transgenic mice, consistent with inhibition of only the mammalian alkyltransferase and maintenance of bacterial alkyltransferase activity in these animals. Thus, whileO6-methylguanine at high concentrations inactivates both bacterial and mammalian alkyltransferases,O6-benzylguanine is a substrate only for the mammalian protein and is unable, perhaps due to steric hindrance, to inhibit the Ada protein.