2 CYTOSOLIC COMPONENTS OF THE HUMAN NEUTROPHIL RESPIRATORY BURST OXIDASE TRANSLOCATE TO THE PLASMA-MEMBRANE DURING CELL ACTIVATION
2 CYTOSOLIC COMPONENTS OF THE HUMAN NEUTROPHIL RESPIRATORY BURST OXIDASE TRANSLOCATE TO THE PLASMA-MEMBRANE DURING CELL ACTIVATION
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DOI：
10.1172/jci114496
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发表时间：
1990-03-01
影响因子：
15.9
通讯作者：
NAUSEEF, WM
中科院分区：
文献类型：
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作者：
CLARK, RA;VOLPP, BD;NAUSEEF, WM
人中性粒细胞的超氧化物形成呼吸爆发氧化酶由膜相关的催化成分和氧化酶激活所需的细胞质成分组成。本研究关注的假设是，当中性粒细胞受到刺激和氧化酶被激活时，细胞质氧化酶成分转移到膜部分。一种多克隆抗血清可识别47和67 kD的两种离散胞质氧化酶组分，用于检测静止和刺激中性粒细胞的电泳膜和胞质组分的转移印迹。与它们在未受刺激的细胞中严格的胞质定位相反，这两种蛋白在被佛波酯和其他刺激激活的中性粒细胞的膜组分中被检测到。这种易位事件是刺激浓度以及暴露于刺激的时间和温度的函数。n -乙基马来酰亚胺阻断了超氧化物的形成，但对2-脱氧葡萄糖没有影响。胞质蛋白的易位与氧化酶的激活之间存在相关性，这是由超氧化物形成确定的。定量分析表明。apprx。在氧化酶的磷酸酯活化过程中，细胞总p47和p67的10%成为膜相关。Percoll密度梯度分数分析表明，这两种蛋白易位的靶膜都是质膜，而不是特异性颗粒或亲氮颗粒的膜。在无细胞氧化酶系统中，证实了花生四烯酮依赖但不依赖膜的胞质氧化酶蛋白沉淀。数据表明，在受刺激的人中性粒细胞中，呼吸爆发氧化酶的激活与47- kd和67-kD细胞质氧化酶组分向质膜的易位密切相关。我们认为这种易位事件在氧化酶活化中很重要。
The superoxide-forming respiratory burst oxidase of human neutrophils is composed of membrane-associated catalytic components and cytosolic constituents required for oxidase activation. This study concerns the hypothesis that cytosolic oxidase components translocate to a membrane fraction when neutrophils are stimulated and the oxidase is activated. A polyclonal antiserum that recognizes two discrete cytosolic oxidase components of 47 and 67 kD was used to probe transfer blots of electrophoresed membrane and cytosol fractions of resting and stimulated neutrophils. In contrast to their strictly cytosolic localization in unstimulated cells, both proteins were detected in membrane fractions of neutrophils activated by phorbol esters and other stimuli. This translocation event was a function of stimulus concentration as well as time and temperature of exposure to the stimulus. It was inhibited by concentrations of N-ethylmaleimide that blocked superoxide formation but was unaffected by 2-deoxyglucose. There was a correlation between translocation of the cytosolic proteins and activation of the oxidase as determined by superoxide formation. Quantitative analyses suggested that .apprx. 10% of total cellular p47 and p67 became membrane-associated during phorbol ester activation of the oxidase. Analysis of Percoll density gradient fractions indicated that the target membrane for translocation of both proteins was the plasma membrane rather than membranes of either specific or azurophilic granules. In the cell-free oxidase system arachidonate-dependent but membrane-independent precipitation of the cytosolic oxidase proteins was demonstrated. The data show that activation of the respiratory burst oxidase in stimulated human neutrophils is closely associated with translocation of the 47- and 67-kD cytosolic oxidase components to the plasma membrane. We suggest that this translocation event is important in oxidase activation.