Hydroxylation of p-nitrophenol by rabbit ethanol-inducible cytochrome P-450 isozyme 3a.
Hydroxylation of p-nitrophenol by rabbit ethanol-inducible cytochrome P-450 isozyme 3a.
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作者：
D. Koop
使用兔肝微粒体和细胞色素 P-450 的六种纯化同工酶研究了对硝基苯酚羟基化为 4-硝基儿茶酚的情况。在 pH 6.8 和 100 microM 对硝基苯酚下微粒体活性最大。在较高底物浓度下观察到抑制作用。在 pH 6.8 和 100 microM 对硝基苯酚下，同工酶 3a 表现出纯化同工酶中最高的活性：比同工酶 6 活性高 3.4 倍，比同工酶 2 和 4 活性高 8 倍。通过添加 4:1 比例的细胞色素 b5/P-450，同工酶 3a 催化的羟基化反应被刺激 2.4 倍。在细胞色素b5的最佳浓度下，同工酶3a的活性比同工酶2和6高8至9倍，比同工酶4高20倍。在相同条件下，同工酶3a催化的丁醇氧化被抑制40%。同工酶 3a 的抗体可抑制未经处理或经乙醇或丙酮处理的兔子微粒体 95% 以上的对硝基苯酚羟化酶活性。微粒体羟化酶活性与同工酶3a的微粒体浓度呈线性相关（相关系数为0.94），且截距接近于零。重构、抗体抑制和相关实验的结果表明同工酶3a是兔微粒体对硝基苯酚羟基化的主要催化剂。乙醇诱导同工酶催化儿茶酚形成的能力对于乙醇增强苯等芳香族化合物的毒性可能很重要。
The hydroxylation of p-nitrophenol to 4-nitrocatechol was investigated using rabbit hepatic microsomes and six purified isozymes of cytochrome P-450. The microsomal activity was maximal at pH 6.8 and at 100 microM p-nitrophenol. At higher substrate concentrations inhibition was observed. At pH 6.8 and 100 microM p-nitrophenol, isozyme 3a exhibited the highest activity of the purified isozymes: 3.4-fold more active than isozyme 6, and 8-fold more active than isozymes 2 and 4. The isozyme 3a-catalyzed hydroxylation reaction was stimulated 2.4-fold by the addition of a 4:1 ratio of cytochrome b5/P-450. At optimal concentrations of cytochrome b5, isozyme 3a was 8- to 9-fold more active than isozymes 2 and 6 and 20-fold more active than isozyme 4. Under the same conditions, isozyme 3a-catalyzed butanol oxidation was inhibited 40%. Antibodies to isozyme 3a inhibited greater than 95% of the p-nitrophenol hydroxylase activity of microsomes from untreated or from ethanol- or acetone-treated rabbits. The microsomal hydroxylase activity was linearly correlated with the microsomal concentration of isozyme 3a (correlation coefficient of 0.94) and had an intercept near zero. The results from reconstitution, antibody inhibition, and correlation experiments indicate that isozyme 3a is the principal catalyst of rabbit microsomal p-nitrophenol hydroxylation. The ability of the ethanol-inducible isozyme to catalyze catechol formation may be important in the ethanol-enhanced toxicity of aromatic compounds such as benzene.