Koshkin V, Lotan O, Pick E
Julius Friedrich Cohnheim-Minerva Center for Phagocyte Research, Department of Human Microbiology, Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978, Israel.
J Biol Chem. 1996 Nov 29;271(48):30326-9. doi: 10.1074/jbc.271.48.30326.
The superoxide (O-2)-generating NADPH oxidase of phagocytes is a multicomponent complex consisting of a membrane-associated flavocytochrome (cytochrome b559), bearing the NADPH binding site and two redox centers (FAD and heme) and three cytosolic activating components: p47(phox), p67(phox), and the small GTPase Rac (1 or 2). The canonical view is that the induction of O-2 generation involves the stimulus-dependent assembly of all three cytosolic components with cytochrome b559, a process mimicked in vitro by a cell-free system activated by anionic amphiphiles. We studied the requirement for individual cytosolic components in the activation of NADPH oxidase in a cell-free system consisting of purified and relipidated cytochrome b559, recombinant p47(phox), p67(phox), and Rac1, and the amphiphile, lithium dodecyl sulfate. We found that pronounced activation of NADPH oxidase can be achieved by exposing cytochrome b559 to p67(phox) and Rac1, in the total absence of p47(phox) (turnover = 60 mol O-2/s/mol cytochrome b559). However, maximal activation (turnover = 153 mol O-2/s/mol cytochrome b559) could only be obtained in the presence of p47(phox). O-2 production, in the absence of p47(phox), was dependent on: high molar ratios of p67(phox) and Rac1 to cytochrome b559, Rac1 being in the GTP-bound form, cytochrome b559 being saturated with FAD, and an optimal concentration of amphiphile. Single cytosolic components or combinations of two cytosolic components, other than p67(phox) and Rac1, were incapable of activation. We conclude that p67(phox) and Rac1 are the only cytosolic components directly involved in the induction of electron transport in cytochrome b559. p47(phox) appears to facilitate or stabilize the interaction of p67(phox) and, possibly, Rac1 with cytochrome b559, and is required for optimal generation of O-2 under physiological conditions.
吞噬细胞中产生超氧化物（O₂⁻）的NADPH氧化酶是一种多组分复合物，由一个膜相关黄素细胞色素（细胞色素b559）组成，其带有NADPH结合位点和两个氧化还原中心（FAD和血红素），以及三个胞质激活成分：p47⁽ᵖʰᵒˣ⁾､p67⁽ᵖʰᵒˣ⁾和小GTP酶Rac（1或2）。传统观点认为，O₂⁻产生的诱导涉及所有三个胞质成分与细胞色素b559的刺激依赖性组装，这一过程在体外可由阴离子两亲物激活的无细胞系统模拟。我们在一个无细胞系统中研究了NADPH氧化酶激活过程中对各个胞质成分的需求，该系统由纯化并重新脂质化的细胞色素b559、重组p47⁽ᵖʰᵒˣ⁾､p67⁽ᵖʰᵒˣ⁾和Rac1，以及两亲物十二烷基硫酸锂组成。我们发现，在完全没有p47⁽ᵖʰᵒˣ⁾的情况下，将细胞色素b559暴露于p67⁽ᵖʰᵒˣ⁾和Rac1可实现NADPH氧化酶的显著激活（周转率 = 60 μmol O₂⁻/s/mol细胞色素b559）。然而，只有在存在p47⁽ᵖʰᵒˣ⁾的情况下才能获得最大激活（周转率 = 153 μmol O₂⁻/s/mol细胞色素b559）。在没有p47⁽ᵖʰᵒˣ⁾的情况下，O₂⁻的产生取决于：p67⁽ᵖʰᵒˣ⁾和Rac1与细胞色素b559的高摩尔比、处于GTP结合形式的Rac1、被FAD饱和的细胞色素b559，以及两亲物的最佳浓度。除了p67⁽ᵖʰᵒˣ⁾和Rac1之外，单个胞质成分或两个胞质成分的组合都无法激活。我们得出结论，p67⁽ᵖʰᵒˣ⁾和Rac1是直接参与细胞色素b559中电子传递诱导的唯一胞质成分。p47⁽ᵖʰᵒˣ⁾似乎促进或稳定了p67⁽ᵖʰᵒˣ⁾以及可能的Rac1与细胞色素b559的相互作用，并且是生理条件下O₂⁻最佳产生所必需的。
