Purification and characterization of oxidoreductases-catalyzing carbonyl reduction of the tobacco-specific nitrosamine 4-methylnitrosamino-1-(3-pyridyl)-1-butanone (NNK) in human liver cytosol
Purification and characterization of oxidoreductases-catalyzing carbonyl reduction of the tobacco-specific nitrosamine 4-methylnitrosamino-1-(3-pyridyl)-1-butanone (NNK) in human liver cytosol
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DOI：
10.1080/00498250050119826
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发表时间：
2000-08-01
期刊：
影响因子：
1.8
通讯作者：
Maser, E
中科院分区：
文献类型：
--
作者：
Atalla, A;Breyer-Pfaff, U;Maser, E
1.从人肝细胞浆中分离纯化了四种酶，证明它们对烟草特有的亚硝胺-4-甲基亚硝胺-1-(3-吡啶)-1-丁酮(NNK)具有还原作用。比较了短链脱氢酶/还原酶(SDR)超家族成员--羰基还原酶(EC 1.1.1.184)与醛酮还原酶(AKR)超家族三种同工酶在酶动力学、共底物依赖性和抑制模式等方面的差异。AKR1C1、1C2和1C4以前被命名为二氢二醇脱氢酶(DD1、DD2和DD4)，其K-m(分别为0.2、0.3和0.8 mM)低于羰基还原酶(7 MM)，而羰基还原酶对NNK的酶效率最高(V-max/K-m)。将酶效率与胞浆中单个酶的相对数量相乘，可以粗略估计它们对总酒精代谢物形成的贡献。这与羰基还原酶的60%、AKR1C1和1C2各20%、AKR1C4.4的1%相似。除AKR1C4外，其余酶对NADPH的选择性强于NADH，NADPH再生系统测定的酶活力最高。甲萘二酮、芦丁和槲皮苷对羧基还原酶活性有广泛的抑制作用，而甲孕酮醋酸酯、酚酞和氟苯那酸对AKR1C1、1C2和1C4.5有较强的抑制作用。综上所述，SDR和AKR超家族的胞浆成员对人体肝脏的还原型NNK解毒有贡献，负责的酶是AKR1C亚家族的羰基还原酶和醛酮还原酶。
1. Four enzymes were purified to homogeneity from human liver cytosol and were demonstrated to be responsible for carbonyl reduction of the tobacco-specific nitrosamine 4-methylnitrosamino-1-(3-pyridyl)-1-butanone (NNK).2. Carbonyl reductase (EC 1.1.1.184), a member of the short-chain dehydrogenase/ reductase (SDR) superfamily, was compared with three isoenzymes of the aldo-keto reductase (AKR) superfamily in terms of enzyme kinetics, co-substrate dependence and inhibition pattern.3. AKR1C1, 1C2 and 1C4, previously designated as dihydrodiol dehydrogenases (DD1, DD2 and DD4), showed lower K-m (0.2, 0.3 and 0.8 mM respectively) than did carbonyl reductase (7 mM), whereas carbonyl reductase exhibited the highest enzyme efficiency (V-max/K-m) for NNK. Multiplication of enzyme efficiencies with the relative quantities of individual enzymes in cytosol resulted in a rough estimate of their contributions to total alcohol metabolite formation. These were similar to 60% for carbonyl reductase, 20% each for AKR1C1 and 1C2, and 1% for AKR1C4.4. Except for AKR1C4, the enzymes had a strong preference for NADPH over NADH, and the highest activities were measured with an NADPH-regenerating system. Carbonyl reductase activity was extensively inhibited by menadione, rutin and quercitrin, whereas medroxyprogesterone acetate, phenolphthalein and flufenamic acid were potent inhibitors of AKR1C1, 1C2 and 1C4.5. In conclusion, cytosolic members of the SDR and AKR superfamilies contribute to reductive NNK detoxification in human liver, the enzymes responsible being carbonyl reductase and aldoketo reductases of the AKR1C subfamily.