Heinemeyer G, Nigam S, Hildebrandt A G
Naunyn Schmiedebergs Arch Pharmacol. 1980 Nov;314(2):201-10. doi: 10.1007/BF00504539.
Cytochrome P-450 dependent oxygenase (3'-hydroxy-hexobarbital) and oxidase activities (hydrogen peroxide) have been measured in hepatic microsomes from guinea pigs, rats and rabbits. A sensitive gas-chromatographic assay was developed to measure the hydroxylated product 3'-hydroxy-hexobarbital. The kinetics of its formation were determined and correlated to hexobarbital type I binding and compared with oxidase activity: in the rat, Vmax for 3'-hydroxyhexobarbital formation was 5.1 and 2.6 nmoles/mg/min, resp. This was increased by phenobarbital treated rabbits, Vmax was 15.0 nmoles/mg/min for hydroxylation and 40.8 for H2O2 formation. Spectral affinity constants (Ks) in control animals were 0.12 mM (rats) and 0.14 mM (rabbits). Phenobarbital treatment decreased these affinity constants, which were similar for each activity measured. In guinea pigs, however, hydroxylation of exobarbital was low (3.1 nmoles/mg/min) and hexobarbital-dependent formation of H2O2 was higher than hydroxylation (Vmax: 7.0 nmoles/mg/min). Phenobarbital treatment led here to two affinity constnts for each activity measured, which however, were alike. The existence of low in addition to high affinity constants observed here might explain the difficulties seen hitherto in correlating hexobarbital binding and metabolism in this species. Total oxidase activity was higher than oxygenase activity in all species tested. It is suggested that oxygenase activity of cytochrome P-450 is not limited by binding but by a competition with oxidase activity for a common intermediary species. This might be peroxy-P-450 (substrate-Fe3+O2(2-), rendering either substrate-Fe3+ O for hydroxylation reaction, or oxidized cytochrome P-450-substrate and hydrogen peroxide as product of oxidase function.
已测定豚鼠、大鼠和家兔肝微粒体中细胞色素P - 450依赖性加氧酶（3'-羟基己巴比妥）和氧化酶活性（过氧化氢）。开发了一种灵敏的气相色谱法来测定羟基化产物3'-羟基己巴比妥。测定了其形成动力学，并与己巴比妥I型结合相关联，并与氧化酶活性进行比较：在大鼠中，3'-羟基己巴比妥形成的Vmax分别为5.1和2.6纳摩尔/毫克/分钟。经苯巴比妥处理的家兔中该值增加，羟基化的Vmax为15.0纳摩尔/毫克/分钟，H2O2形成的Vmax为40.8纳摩尔/毫克/分钟。对照动物的光谱亲和常数（Ks）分别为0.12毫摩尔（大鼠）和0.14毫摩尔（家兔）。苯巴比妥处理降低了这些亲和常数，所测的每种活性的亲和常数相似。然而，在豚鼠中，异戊巴比妥的羟基化作用较低（3.1纳摩尔/毫克/分钟），己巴比妥依赖性H2O2的形成高于羟基化作用（Vmax：7.0纳摩尔/毫克/分钟）。苯巴比妥处理导致所测的每种活性有两个亲和常数，但二者相似。此处观察到除高亲和常数外还存在低亲和常数，这可能解释了迄今为止在该物种中关联己巴比妥结合与代谢时遇到的困难。在所有测试物种中，总氧化酶活性高于加氧酶活性。有人提出，细胞色素P - 450的加氧酶活性不受结合的限制，而是受与氧化酶活性竞争共同中间物种的限制。这可能是过氧 - P - 450（底物 - Fe3 + O2(2 - )），它要么提供底物 - Fe3 + O用于羟基化反应，要么生成氧化型细胞色素P - 450 - 底物和过氧化氢作为氧化酶功能的产物。
