Activation of the respiratory burst enzyme from human neutrophils in a cell-free system. Evidence for a soluble cofactor.
Activation of the respiratory burst enzyme from human neutrophils in a cell-free system. Evidence for a soluble cofactor.
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在无细胞系统中激活人中性粒细胞的呼吸爆发酶。
DOI：
10.1172/jci111884
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发表时间：
1985
期刊：
影响因子：
--
通讯作者：
Snyderman,R
中科院分区：
文献类型：
--
作者：
McPhail,LC;Shirley,PS;Clayton,CC;Snyderman,R
吞噬细胞中呼吸爆发的激活是一个重要的宿主防御过程，目前还没有很好的理解。我们现在报告的发展，无细胞系统的激活NADPH氧化酶，呼吸爆发酶，在人类中性粒细胞。通过将花生四烯酸添加到来自破坏的未刺激的细胞（无花生四烯酸，0.2;用花生四烯酸，3.4 nmol超氧阴离子/min/mg）的核后上清液（500 g）中来实现激活，并且取决于花生四烯酸的浓度和存在的细胞材料的量。根据NADPH的米氏常数为32 μ M和最适pH为7.0-7.5，花生四烯酸刺激的活性似乎是NADPH氧化酶。通过高速离心分离500-g上清液揭示了对可溶性和颗粒状辅因子的需要。花生四烯酸对NADPH氧化酶的激活在来自未刺激细胞的高速沉淀部分中没有发生，但可以通过添加高速上清液来恢复。此外，用低浓度的化学引诱物N-甲酰基-甲硫氨酰-亮氨酰-苯丙氨酸或肿瘤促进剂佛波醇肉豆蔻酸乙酸酯引发完整的中性粒细胞，取代了在高速颗粒中花生四烯酸盐激活NADPH氧化酶所需的可溶性因子。这种无细胞系统现在可以用来提供进一步的了解引发的生化基础和参与NADPH氧化酶激活的终端机制。
Activation of the respiratory burst in phagocytic cells, an important host defense process, is not yet well understood. We now report the development of a cell-free system for activation of NADPH oxidase, the respiratory burst enzyme, in human neutrophils. Activation was achieved by the addition of arachidonic acid to a postnuclear supernatant (500 g) from disrupted unstimulated cells (no arachidonate, 0.2; with arachidonate, 3.4 nmol superoxide anion/min per mg) and was dependent on both the concentration of arachidonate and on the amount of cellular material present. Activity stimulated by arachidonate appeared to be NADPH oxidase based on a Michaelis constant for NADPH of 32 microM and a pH optimum of 7.0-7.5. Separation of the 500-g supernatant by high speed centrifugation revealed a requirement for both soluble and particulate cofactors. Activation of NADPH oxidase by arachidonate did not occur in the high speed pellet fraction from unstimulated cells but could be restored by the addition of the high speed supernatant. In addition, priming of intact neutrophils with low concentrations of the chemoattractant N-formyl-methionyl-leucyl-phenylalanine or the tumor promoter phorbol myristate acetate replaced the soluble factor requirement for NADPH oxidase activation by arachidonate in the high speed pellet. This cell-free system can now be used to provide further insight into the biochemical basis of priming and the terminal mechanisms involved in the activation of NADPH oxidase.