ONE-ELECTRON AND 2-ELECTRON REDUCTION OF 2-METHYL-1,4-NAPHTHOQUINONE BIOREDUCTIVE ALKYLATING-AGENTS - KINETIC-STUDIES, FREE-RADICAL PRODUCTION, THIOL OXIDATION AND DNA-STRAND-BREAK FORMATION
ONE-ELECTRON AND 2-ELECTRON REDUCTION OF 2-METHYL-1,4-NAPHTHOQUINONE BIOREDUCTIVE ALKYLATING-AGENTS - KINETIC-STUDIES, FREE-RADICAL PRODUCTION, THIOL OXIDATION AND DNA-STRAND-BREAK FORMATION
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DOI：
10.1042/bj3010021
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发表时间：
1994-07-01
影响因子：
4.1
通讯作者：
CADENAS, E
中科院分区：
文献类型：
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作者：
GIULIVI, C;CADENAS, E
分别用纯化的NADPH-细胞色素P-450还原酶和DT-黄递酶研究了生物还原烷基化试剂2-甲氧基萘酚和2-氯甲基萘醌的单电子和双电子还原反应，并从动力学常数、氧自由基产生、硫醇氧化和DNA链断裂形成等方面进行了表征。催化中心活性结果表明，DT-黄递酶对苯二酚I的催化活性远高于NADPH-细胞色素P-450还原酶，但两种酶的K-m值相近(1.2-3.0µM)。单电子转移黄素酶也催化了对苯二酚的还原，但DT-黄递酶对这种对苯二酚的行为不允许计算动力学常数。由这些对苯二酚的单电子和双电子催化引起的氧化还原转变的一个显著特征是歧化反应和自氧化反应的不同贡献。在前一种情况下，大约26%的NADPH消耗是自氧化(以H_2O_2的形成)，而在后一种情况下，自氧化成分占NADPH消耗的大部分(98%)。这种差异被超氧化物歧化酶消除，在NADPH-细胞色素P-450催化过程中，超氧化物歧化酶将自氧化作用增强到最大值。E.S.R.分析表明，超氧阴离子自由基的产生被超氧化物歧化酶抑制，而不受过氧化氢酶的影响。在GSH存在下，这些对苯二酚的单电子和双电子还原伴随着硫基自由基的形成。虽然超氧化物歧化酶抑制了硫醇自由基的产生。在这两种情况下，该酶在NADPH-细胞色素P-450催化对苯二酚I的过程中促进GSSG的积累，而在DT-黄递酶对对苯二酚的还原过程中抑制GSSG的形成。苯二酚I的单电子和双电子还原导致小牛胸腺DNA链断裂形成，这一过程(A)在透析的DNA和Desferal存在的情况下大大减少，(B)对超氧化物歧化酶和/或过氧化氢酶部分敏感。这些发现是合理的，因为金属离子连接到DNA上，保护了在酶还原苯醌过程中产生的超氧阴离子的毒性效应。
The one- and two-electron enzymic reduction of the bioreductive alkylating agents 2-methylmethoxynaphthoquinone (quinone I) and 2-chloromethylnaphthoquinone (quinone II) was studied with purified NADPH-cytochrome P-450 reductase and DT-diaphorase respectively, and characterized in terms of kinetic constants, oxyradical production, thiol oxidation and DNA-strand-break formation. The catalytic-centre activity values indicated that DT-diaphorase catalysed the reduction of quinone I far more efficiently than NADPH-cytochrome P-450 reductase, although the K-m values of the two enzymes for this quinone were similar (1.2-3.0 mu M). The one-electron-transfer flavoenzyme also catalysed the reduction of quinone II, but the behaviour of DT-diaphorase towards this quinone did not permit calculation of kinetic constants. A salient feature of the redox transitions caused by the one- and two-electron catalysis of these quinones was the different contributions of disproportionation and autoxidation reactions respectively. In the former case, about 26 % of NADPH consumed was accounted for in terms of autoxidation (as H2O2 formation), whereas in the latter, the autoxidation component accounted for most (98 %) of the NADPH consumed. This difference was abrogated by superoxide dismutase, which enhanced autoxidation during NADPH-cytochrome P-450 catalysis to a maximal value. E.s.r. analysis indicated the formation of superoxide radicals, the signal of which was suppressed by superoxide dismutase and unaffected by catalase. The one- and two-electron reduction of these quinones in the presence of GSH was accompanied by formation of thiyl radicals. Although superoxide dismutase suppressed the thiol radical e.s.r. signal in both instances, the enzyme enhanced GSSG accumulation during NADPH-cytochrome P-450 catalysis of quinone I, whereas it inhibited GSSG formation during reduction of the quinone by DT-diaphorase. One- and two-electron reduction of quinone I led to calf thymus DNA-strand-break formation, a process that (a) was substantially decreased in experiments performed with dialysed DNA and in the presence of desferal and (b) was partially sensitive to superoxide dismutase and/or catalase. These findings are rationalized in terms of the occurrence of metal ions ligated to DNA, protecting against the toxic effects of superoxide radicals generated during enzymic reduction of quinones.