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首页> 外文学位 >Studies on competitive interactions between cytochromes P450 2A6 and 2E1 for NADPH-cytochrome P450 oxidoreductase in the microsomal membranes produced by a baculovirus expression system.
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Studies on competitive interactions between cytochromes P450 2A6 and 2E1 for NADPH-cytochrome P450 oxidoreductase in the microsomal membranes produced by a baculovirus expression system.

机译：研究杆状病毒表达系统产生的微粒体膜中细胞色素P450 2A6和2E1对NADPH-细胞色素P450氧化还原酶的竞争性相互作用。

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摘要

The propose of the present study is to develop an expression system to coexpress human cytochromes P450 (P450) and human NADPH:cytochrome oxidoreductase (hOR) in the Spodoptera frugiperda cells (Sf 9 cells) and to use this system to investigate the interactions between P450 enzymes and OR in the microsomal membranes. Microsomes containing human cytochrome P450 2A6 (h2A6) coexpressed with human OR (hOR) via a baculovirus expression system displayed coumarin hydroxylase activity with apparent K{dollar}rmsb{lcub}m{rcub}{dollar} and V{dollar}rmsb{lcub}max{rcub}{dollar} values of 0.41 {dollar}mu{dollar}M and 4.05 nmol/min/nmol P450, respectively. Incorporation of purified rat liver cytochrome b{dollar}sb5{dollar} (b{dollar}sb5{dollar}) into the microsomes increased the V{dollar}rmsb{lcub}max{rcub}{dollar} 2.5-fold, but did not affect the K{dollar}rmsb{lcub}m{rcub}{dollar}. The N-nitrosodimethylamine (NDMA) demethylase activity of human cytochrome P450 2E1 (h2E1) coexpressed similarly was characterized previously. Coumarin was shown not to be a substrate nor an inhibitor of h2E1, and NDMA was not a substrate nor an inhibitor of h2A6. In microsomes containing h2A6, h2E1, and hOR (M-h2A6-h2E1-hOR) obtained from a triple expression system, the two P450 enzymes were shown to compete with each other for interaction with hOR. In incubations with M-h2A6-h2E1-hOR, the presence of a h2A6 substrate (coumarin) decreased NDMA demethylase activity by a maximum of 47%, and the presence of a h2E1 substrate (NDMA) decreased coumarin hydroxylase activity by a maximum of 19%. This substrate-induced competition between h2A6 and h2E1 was decreased by the addition of purified b{dollar}sb5{dollar}. In the absence of a substrate, the NADPH-dependent H{dollar}sb2{dollar}O{dollar}sb2{dollar} formation was high in both M-h2A6-h2E1-hOR and M-h2E1-hOR, but low in M-h2A6-hOR. The addition of NDMA had little effect on the H202 formation in M-h2A6-h2E1-hOR and M-h2E1-hOR. The addition of coumarin, however, slightly decreased H{dollar}sb2{dollar}O{dollar}sb2{dollar} formation in M-h2A6-h2E1-hOR, but drastically increased H{dollar}sb2{dollar}O{dollar}sb2{dollar} formation in M-h2A6-hOR. These results suggest that the presence of a h2A6 substrate decreased the electron flow to h2E1 in M-2A6-h2E1-hOR. The activities of coumarin hydroxylase and NDMA demethylase of M-h2A6-h2E1-hOR were decreased and increased, respectively, by an increase in ionic strength. The ionic strength, however, did not drastically change the substrate-induced competition between h2A6 and h2E1 for hOR. The results demonstrate the usefulness of the co-expression system for mechanistic studies and illustrate that the interaction of monooxygenase enzymes in the microsomal membrane is regulated by the presence of substrates and b{dollar}sb5{dollar}.
机译：本研究的目的是开发一种表达系统，以在草地贪夜蛾细胞（Sf 9细胞）中共表达人类细胞色素P450（P450）和人类NADPH：细胞色素氧化还原酶（hOR），并使用该系统研究P450之间的相互作用体膜中的酶和或。通过杆状病毒表达系统与人OR（hOR）共表达的含有人细胞色素P450 2A6（h2A6）的微粒体显示出香豆素羟化酶活性，并具有明显的K {dollar} rmsb {lcub} m {rcub} {dollar}和V {dollar} rmsb {lcub } max {rcub} {dollar}值分别为0.41 {dollar} mu {dollar} M和4.05 nmol / min / nmol P450。将纯化的大鼠肝细胞色素b {dollar} sb5 {dollar}（b {dollar} sb5 {dollar}）掺入微粒体可使V {dollar} rmsb {lcub} max {rcub} {dollar}增加2.5倍，但不会影响K {dollar} rmsb {lcub} m {rcub} {dollar}。先前已经表征了共表达相似地共表达的人细胞色素P450 2E1（h2E1）的N-亚硝基二甲胺（NDMA）脱甲基酶活性。香豆素被证明不是h2E1的底物或抑制剂，而NDMA不是h2A6的底物或抑制剂。从三重表达系统获得的含有h2A6，h2E1和hOR（M-h2A6-h2E1-hOR）的微粒体中，两种P450酶显示彼此竞争与hOR的相互作用。与M-h2A6-h2E1-hOR孵育时，h2A6底物（香豆素）的存在使NDMA脱甲基酶活性最多降低47％，而h2E1底物（NDMA）的存在使香豆素羟化酶活性最多降低19％ ％。通过加入纯化的b {dollar} sb5 {dollar}，可以降低h2A6和h2E1之间这种底物诱导的竞争。在没有底物的情况下，NADPH依赖的H {dollar} sb2 {dollar} O {dollar} sb2 {dollar}的形成在M-h2A6-h2E1-hOR和M-h2E1-hOR中均较高，但M -h2A6-hOR。 NDMA的添加对M-h2A6-h2E1-hOR和M-h2E1-hOR中H202的形成几乎没有影响。然而，香豆素的添加在M-h2A6-h2E1-hOR中略微减少了H {dolb} sb2 {dollar} O {dollar} sb2 {dollar}的形成，但显着增加了H {dollar} sb2 {dollar} O {dollar} M-h2A6-hOR中sb2 {dollar}的形成。这些结果表明，在M-2A6-h2E1-hOR中，h2A6底物的存在降低了流向h2E1的电子。 M-h2A6-h2E1-hOR的香豆素羟化酶和NDMA脱甲基酶的活性分别通过离子强度的增加而降低和增加。然而，离子强度并没有显着改变h2A6和h2E1之间的底物诱导的hOR竞争。结果证明了共表达系统对于机理研究的有用性，并表明底物和b {dollar} sb5 {dollar}的存在调节了微粒体膜中单加氧酶的相互作用。

著录项

  • 作者

    Tan, Yizheng.;

  • 作者单位

    Rutgers The State University of New Jersey - New Brunswick.;

  • 授予单位 Rutgers The State University of New Jersey - New Brunswick.;
  • 学科 Biology Molecular.; Biology Cell.; Chemistry Biochemistry.
  • 学位 Ph.D.
  • 年度 1997
  • 页码 82 p.
  • 总页数 82
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 分子遗传学;细胞生物学;生物化学;
  • 关键词

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