Iyanagi T, Suzaki T, Kobayashi S
J Biol Chem. 1981 Dec 25;256(24):12933-9.
The spectral changes of cytochrome P-450 associated with mixed-function oxidation of hexobarbital and aminopyrine were investigated in perfused rat liver, using reflectance spectrophotometry. Simultaneously, the oxidation-reduction state of pyridine nucleotide(s) and oxygen uptake were measured from the same liver. Difference spectra were observed after infusion of hexobarbital or aminopyrine. Spectra obtained from livers of fasted, sodium phenobarbital-treated rats were similar to those of cytochrome P-450 . substrate complex (type I); the spectrum from fed, sodium phenobarbital-treated rats was a mixture of substrate . cytochrome P-450 complex and reduced (an oxygenated) cytochrome P-450. In fed, sodium phenobarbital-treated rats, the steady state level of reduced (an oxygenated) cytochrome P-450 was dependent on the concentration and the kind of substrates added, and the level correlated well with mixed-function oxidase activity. In the absence of exogenous substrates, the degree of the formation of reduced (an oxygenated) cytochrome P-450 was small. These results indicate that mixed-function oxidase activity in the intact cell is regulated by substrate-binding to cytochrome P-450. In fasted, sodium phenobarbital-treated rats, a substantial oxidation of pyridine nucleotide was observed in the presence of hexobarbital. The increase of oxygen uptake was a 2- to 2.5-fold smaller rate than in livers from fed, sodium phenobarbital-treated rats. Infusion of sorbitol (2 mM), a glycogenic substrate in fasted rats, stimulated oxygen uptake about 3-fold. Furthermore, reduced (an oxygenated) cytochrome P-450 increased in the presence of sorbitol. These results indicate that the rate of NADPH supply is rate-limiting for mixed-function oxidation in livers of fasted, sodium phenobarbital-treated rats. Finally, the difference spectrum of cytochrome P-450, the surface fluorescence of pyridine nucleotide, and the oxygen uptake were compared using livers from untreated, alloxan-treated and sodium phenobarbital-treated rats.
采用反射分光光度法，在灌注大鼠肝脏中研究了与己巴比妥和氨基比林混合功能氧化相关的细胞色素P-450的光谱变化。同时，从同一肝脏中测量吡啶核苷酸的氧化还原状态和氧摄取。注入己巴比妥或氨基比林后观察到差异光谱。从禁食的、苯巴比妥钠处理的大鼠肝脏获得的光谱类似于细胞色素P-450·底物复合物（I型）的光谱；从喂食的、苯巴比妥钠处理的大鼠肝脏获得的光谱是底物·细胞色素P-450复合物和还原型（氧化型）细胞色素P-450的混合物。在喂食的、苯巴比妥钠处理的大鼠中，还原型（氧化型）细胞色素P-450的稳态水平取决于添加底物的浓度和种类，且该水平与混合功能氧化酶活性密切相关。在没有外源底物的情况下，还原型（氧化型）细胞色素P-450的形成程度较小。这些结果表明，完整细胞中的混合功能氧化酶活性受底物与细胞色素P-450结合的调节。在禁食的、苯巴比妥钠处理的大鼠中，在己巴比妥存在下观察到吡啶核苷酸的大量氧化。氧摄取的增加速率比喂食的、苯巴比妥钠处理的大鼠肝脏中的氧摄取增加速率小2至2.5倍。注入山梨醇（2 mM），一种禁食大鼠中的糖原生成底物，可刺激氧摄取约3倍。此外，在山梨醇存在下还原型（氧化型）细胞色素P-450增加。这些结果表明，在禁食的、苯巴比妥钠处理的大鼠肝脏中，NADPH供应速率是混合功能氧化的限速因素。最后，使用未处理的、四氧嘧啶处理的和苯巴比妥钠处理的大鼠肝脏比较了细胞色素P-450的差异光谱、吡啶核苷酸的表面荧光和氧摄取。
