NAD(P)H oxidase activity in human neutrophils stimulated by phorbol myristate acetate.
NAD(P)H oxidase activity in human neutrophils stimulated by phorbol myristate acetate.
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佛波醇肉豆蔻酸酯乙酸酯刺激人中性粒细胞中的 NAD(P)H 氧化酶活性。
DOI：
10.1172/jci109994
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发表时间：
1980
期刊：
影响因子：
--
通讯作者：
Lehrer,RI
中科院分区：
文献类型：
--
作者：
Suzuki,Y;Lehrer,RI
佛波醇肉豆蔻酸酯在正常人中性粒细胞中激活一种单一的酶实体，该实体在未刺激的细胞中处于休眠状态，在pH 7.0时最佳活性，并且能够氧化NADH或NADPH，产生NAD（P）+和超氧化物（O27）。比较荧光和分光光度测量支持化学计量NAD（P）H + 20（2）导致NAD（P）+ + 20（27）+ H+。在pH5.5和6.0时，NAD（P）+的产生似乎相当可观，这主要是由于NAD（P）H通过由HO 27（过羟基自由基）（O 27的共轭酸）引发的链式反应的非酶促氧化。这种人为因素导致早期错误地指定了NAD（P）H氧化酶的最佳酸性pH值，但通过在荧光测定中加入超氧化物歧化酶可以防止这种人为因素。NAD（P）H氧化酶对NADPH（Km = 0.15 +/- 0.03 mM）的活性高于对NADH（Km = 0.68 +/- 0.2 mM）的活性。没有发现氧化酶活性受NAD（P）H的变构调节。注意到佛波醇肉豆蔻酸酯乙酸酯诱导的O27产生在完整的中性粒细胞中受pH调节，表明NAD（P）H氧化酶定位于质膜中，其活性可能受到局部H+浓度的（自动）调节。
Phorbol myristate acetate activated in normal human neutrophils a single enzymatic entity that was dormant in unstimulated cells, optimally active at pH 7.0, and capable of oxidizing either NADH or NADPH, producing NAD(P)+ and superoxide (O27). Comparative fluorometric and spectrophotometric measurements supported the stoichiometry NAD(P)H + 20(2) leads to NAD(P)+ + 20(27) + H+. the seemingly considerable NAD(P)+ production at pH 5.5 and 6.0 was due largely to nonenzymatic oxidation of NAD(P)H by chain reactions initiated by HO27 (perhydroxyl radical), the conjugate acid of O27. This artifact, responsible for earlier erroneous assignments of an acid pH optimum for NAD(P)H oxidase, was prevented by including superoxide dismutase in fluorometric assays. NAD(P)H oxidase was more active towards NADPH (Km = 0.15 +/- 0.03 mM) than NADH (Km = 0.68 +/- 0.2 mM). No suggestion that oxidase activity was allosterically regulated by NAD(P)H was seen. Phorbol myristate acetate-induced O27 production was noted to be modulated by pH in intact neutrophils, suggesting that NAD(P)H oxidase is localized in the plasma membrane where its activity may be subject to (auto) regulation by local H+ concentrations.