Ječmen Tomáš, Ptáčková Renata, Kavan Daniel, Cerná Věra, Hodek Petr, Stiborová Marie, Hudeček Jiří, Sulc Miroslav
Neuro Endocrinol Lett. 2014;35 Suppl 2:114-22.
The mammalian mixed function oxidase (MFO) system participates in hydroxylation of many hydrophobic endogenous compounds as well as xenobiotics such as drugs and carcinogens. This biotransformation system, located in a membrane of endoplasmic reticulum, consists of cytochrome P-450 (P450), NADPH:P450 oxidoreductase and a facultative component, cytochrome b5. The knowledge of the interactions among the individual components of the MFO system is essential to understand the relationships between the structure and function of this system that finally dictate a qualitative and quantitative pattern of produced metabolites (e.g. detoxified xenobiotics and/or activated carcinogens). To elucidate the quantitative aspects of the interactions within the MFO system we acquired the photo-initiated cross-linking approach.
The photo-initiated cross-linking employing cytochrome b5 as a protein nanoprobe [an amino acid analogue of methionine (pMet) was incorporated into cytochrome b5 sequence during recombinant expression] was used to quantify its interaction with P450 2B4 in a functional membrane complex. The cross-linking was initiated by UV-irradiation that formed from a pMet photolabile diazirine group highly reactive carbene biradical. This biradical is able to covalently bind amino acids in the close proximity and to form cross-link. The Met 96 of cytochrome b5 is situated in a linker region between its catalytic and membrane domains, while Met 126 and 131 are located in its membrane domain. The combination of several methods (electrophoresis in polyacrylamide gel, isoelectric focusing, Edman N-terminal degradation and amino acid analysis) was employed to characterize the molar ratio of P450 2B4 to cytochrome b5 in formed covalent cross-links to quantify their transient interactions.
The successfully produced cytochrome b5 nanoprobe (with confirmed pMet incorporation by mass spectrometry) stimulates the catalytical activity of P450 2B4 when reconstituted with NADPH:P450 oxidoreductase in vitro in dilauroylphosphatidylcholine (DLPC) vesicles. The cross-linking was carried out in similar reconstituted system without NADPH:P450 oxidoreductase, and at least three products were separated on 1D SDS-PAGE. The molar ratio of P450 to cytochrome b5 in each complex was estimated using the above-mentioned combination of methods as 1:1, 1:2 and 2:1.
The results demonstrate the utility of cytochrome b5 nanoprobe to study the interactions in MFO system. Using this nanoprobe, heterodimer with P450 2B4 and in addition also heterooligomers were identified, suggesting rather complex interactions of both proteins in this system that suppose the formation of such multimeric structures in the membrane of endoplasmic reticulum.
哺乳动物混合功能氧化酶（MFO）系统参与许多疏水性内源性化合物以及诸如药物和致癌物等外源性物质的羟基化反应。这个生物转化系统位于内质网的膜上，由细胞色素P - 450（P450）、NADPH：P450氧化还原酶和一个兼性成分细胞色素b5组成。了解MFO系统各个组分之间的相互作用对于理解该系统的结构与功能之间的关系至关重要，而这种关系最终决定了所产生代谢物（如解毒的外源性物质和/或活化的致癌物）的定性和定量模式。为了阐明MFO系统内相互作用的定量方面，我们采用了光引发交联方法。
使用细胞色素b5作为蛋白质纳米探针的光引发交联（在重组表达过程中将甲硫氨酸的氨基酸类似物（pMet）掺入细胞色素b5序列中）来定量其与功能性膜复合物中P450 2B4的相互作用。交联反应由紫外线照射引发，紫外线由pMet光不稳定的重氮基团形成高反应性的卡宾双自由基。这个双自由基能够共价结合附近的氨基酸并形成交联。细胞色素b5的甲硫氨酸96位于其催化结构域和膜结构域之间的连接区域，而甲硫氨酸126和131位于其膜结构域。采用几种方法（聚丙烯酰胺凝胶电泳、等电聚焦、埃德曼N端降解和氨基酸分析）的组合来表征形成的共价交联中P450 2B4与细胞色素b5的摩尔比，以定量它们的瞬时相互作用。
成功制备的细胞色素b5纳米探针（通过质谱确认了pMet的掺入）在体外与NADPH：P450氧化还原酶一起在二月桂酰磷脂酰胆碱（DLPC）囊泡中重构时，刺激了P450 2B4的催化活性。交联反应在没有NADPH：P450氧化还原酶的类似重构系统中进行，并且在一维SDS - PAGE上分离出至少三种产物。使用上述方法组合估计每个复合物中P450与细胞色素b5的摩尔比为1:1、1:2和2:1。
结果证明了细胞色素b5纳米探针在研究MFO系统相互作用中的实用性。使用这种纳米探针，鉴定出了与P450 2B4的异二聚体以及异寡聚体，这表明该系统中两种蛋白质的相互作用相当复杂，推测在内质网膜中形成了这种多聚体结构。
