Shaw P M, Hosea N A, Thompson D V, Lenius J M, Guengerich F P
PanVera Corporation, Madison, Wisconsin 53711, USA.
Arch Biochem Biophys. 1997 Dec 1;348(1):107-15. doi: 10.1006/abbi.1997.0378.
The development of enzyme and buffer premixes for in vitro biotransformation assays is described. The protein premixes contain a mixture of three recombinant human proteins, cytochrome P450 (P450) 3A4, NADPH-P450 reductase, cytochrome b5, and liposomes. The buffer premix contains reagents which, when diluted, provide for optimal metabolic activity with selected P450 3A4 substrates. P450 3A4 premixes were competent in the oxidation of known substrates including testosterone, midazolam, nifedipine, erythromycin, benzphetamine, and amitriptyline. Premixes stored at -80 degrees C for 2 months and those that underwent an additional five freeze/thaw cycles were able to hydroxylate testosterone at turnover rates similar to freshly prepared reconstitution mixes. In addition, premixes stored unfrozen at 4 degrees C for 2 weeks showed no significant loss in the rate of testosterone 6 beta-hydroxylation by P450 3A4. Premixes prepared with and without reduced glutathione, a component which had previously been found to be important for P450 3A4 reactions, were equally efficient at carrying out testosterone hydroxylation under these conditions. Kinetic parameters determined for the metabolism of testosterone, amitriptyline, nifedipine, and benzphetamine using P450 3A4 premixes were compared with human pooled microsomes and insect microsomes prepared from cells infected with a baculovirus containing two cDNA inserts coding for P450 3A4 and NADPH-P450 reductase. Each format gave different Vmax and K(m) values indicating different catalytic efficiencies. Analysis of P450 1A2 premixes which contained different lipid concentrations indicated that Vmax and K(m) could be altered. The availability of human P450 recombinant enzymes and the development of the P450 premixes that remain active after being stored frozen should allow for rapid identification of novel P450 substrates and inhibitors and the development of large-scale screening assays.
本文描述了用于体外生物转化分析的酶和缓冲液预混物的开发。蛋白质预混物包含三种重组人蛋白的混合物，即细胞色素P450（P450）3A4、NADPH - P450还原酶、细胞色素b5和脂质体。缓冲液预混物包含一些试剂，稀释后能为选定的P450 3A4底物提供最佳代谢活性。P450 3A4预混物能够氧化包括睾酮、咪达唑仑、硝苯地平、红霉素、苄非他明和阿米替林在内的已知底物。在 - 80℃下储存2个月的预混物以及经历额外五次冻融循环的预混物，能够以与新鲜制备的重构混合物相似的周转速率使睾酮羟化。此外，在4℃下未冷冻储存2周的预混物，P450 3A4对睾酮6β - 羟化的速率没有显著损失。在有或没有还原型谷胱甘肽（先前发现该成分对P450 3A4反应很重要）的情况下制备的预混物，在这些条件下进行睾酮羟化的效率相同。使用P450 3A4预混物测定的睾酮、阿米替林、硝苯地平和苄非他明代谢的动力学参数，与从感染含有编码P450 3A4和NADPH - P450还原酶的两个cDNA插入片段的杆状病毒的细胞制备的人混合微粒体和昆虫微粒体进行了比较。每种形式给出了不同的Vmax和Km值，表明催化效率不同。对含有不同脂质浓度的P450 1A2预混物的分析表明，Vmax和Km可以改变。人P450重组酶的可用性以及冷冻储存后仍保持活性的P450预混物的开发，应有助于快速鉴定新型P450底物和抑制剂，并开发大规模筛选分析方法。
