Constitutive NADPH-dependent electron transferase activity of the Nox4 dehydrogenase domain.
Constitutive NADPH-dependent electron transferase activity of the Nox4 dehydrogenase domain.
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DOI：
10.1021/bi9022285
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发表时间：
2010-03-23
期刊：
影响因子：
2.9
通讯作者：
Lambeth, J. David
中科院分区：
文献类型：
--
作者：
Nisimoto, Yukio;Jackson, Heather M.;Ogawa, Hisamitsu;Kawahara, Tsukasa;Lambeth, J. David
NADPH氧化酶4（Nox 4）是组成型活性的，而Nox 2需要胞质调节亚基p47phox和p67phox以及由佛波醇12-肉豆蔻酸酯13-乙酸酯（PMA）活化的活化Rac。进行这项研究以鉴定赋予组成型活性的Nox 4上的结构域。来自Nox 4表达细胞的裂解物表现出组成型NADPH依赖性而非NADH依赖性过氧化氢产生，NADPH的Km为55 ± 10 μ M。使用蛋白质印迹法估计细胞裂解物中Nox 4的浓度，并计算出200 mol H2O2 min − 1（mol Nox 4）− 1的周转率。由Nox 2跨膜（TM）结构域和Nox 4脱氢酶（DH）结构域组成的嵌合蛋白（Nox 2/4）在不存在胞质调节亚基的情况下显示出H2O2产生。相反，嵌合体Nox 4/2，由Nox 4 TM和Nox 2 DH结构域组成，表现出PMA依赖性激活，需要调节亚基的共表达。纯化来自几种Nox同种型的Nox DH结构域并评估其电子转移酶活性。Nox 1 DH、Nox 2 DH和Nox 5 DH结构域对人工电子受体表现出几乎检测不到的活性，而Nox 4 DH结构域对细胞色素c（160 min − 1，主要不依赖于超氧化物歧化酶）、铁氰化物（470 min − 1）和其他电子受体（人工染料和细胞色素b5）表现出显著的还原速率。速率与细胞裂解物中Nox 4全酶产生H2O2所观察到的速率相似。该活性需要添加FAD，并且在NADPH而不是NADH的情况下观察到。这些结果表明，Nox4 DH结构域存在于一个固有的激活状态，从NADPH到FAD的电子转移很可能是限速的NADPH依赖性还原氧的holo-Nox4。
NADPH oxidase 4 (Nox4) is constitutively active, while Nox2 requires the cytosolic regulatory subunits p47phox and p67phox and activated Rac with activation by phorbol 12-myristate 13-acetate (PMA). This study was undertaken to identify the domain on Nox4 that confers constitutive activity. Lysates from Nox4-expressing cells exhibited constitutive NADPH- but not NADH-dependent hydrogen peroxide production with a Km for NADPH of 55 ± 10 μM. The concentration of Nox4 in cell lysates was estimated using Western blotting and allowed calculation of a turnover of ∼200 mol of H2O2 min−1 (mol of Nox4)−1. A chimeric protein (Nox2/4) consisting of the Nox2 transmembrane (TM) domain and the Nox4 dehydrogenase (DH) domain showed H2O2 production in the absence of cytosolic regulatory subunits. In contrast, chimera Nox4/2, consisting of the Nox4 TM and Nox2 DH domains, exhibited PMA-dependent activation that required coexpression of regulatory subunits. Nox DH domains from several Nox isoforms were purified and evaluated for their electron transferase activities. Nox1 DH, Nox2 DH, and Nox5 DH domains exhibited barely detectable activities toward artificial electron acceptors, while the Nox4 DH domain exhibited significant rates of reduction of cytochrome c (160 min−1, largely superoxide dismutase-independent), ferricyanide (470 min−1), and other electron acceptors (artificial dyes and cytochrome b5). Rates were similar to those observed for H2O2 production by the Nox4 holoenzyme in cell lysates. The activity required added FAD and was seen with NADPH but not NADH. These results indicate that the Nox4 DH domain exists in an intrinsically activated state and that electron transfer from NADPH to FAD is likely to be rate-limiting in the NADPH-dependent reduction of oxygen by holo-Nox4.
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