Cell-free translocation of recombinant p47-phox, a component of the neutrophil NADPH oxidase: effects of guanosine 5'-O-(3-thiotriphosphate), diacylglycerol, and an anionic amphiphile.
Cell-free translocation of recombinant p47-phox, a component of the neutrophil NADPH oxidase: effects of guanosine 5'-O-(3-thiotriphosphate), diacylglycerol, and an anionic amphiphile.
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重组 p47-phox（中性粒细胞 NADPH 氧化酶的一个组成部分）的无细胞易位：鸟苷 5-O-（3-硫代三磷酸）、二酰基甘油和阴离子两亲物的作用。
DOI：
10.1021/bi00125a017
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发表时间：
1992
期刊：
影响因子：
2.9
通讯作者：
Lambeth,JD
中科院分区：
文献类型：
--
作者：
Tyagi,SR;Neckelmann,N;Uhlinger,DJ;Burnham,DN;Lambeth,JD
1991年12月20日收到的修订版摘要：我们以前报道过，二酰基甘油（diC 8）和GTP 7S与阴离子两亲物如十二烷基硫酸钠（SDS）协同作用，在由中性粒细胞质膜和细胞溶质组成的无细胞系统中产生高速率的超氧化物[Burnham，D. N.，Uhlinger，D. J.，&兰贝斯，J. D.（1990）J.Biol.Chem.265，17550-17559]。在这里，我们研究了这些激活因子在体外翻译的放射性标记的重组p47-phox蛋白质膜协会的影响。通过与质膜共沉降测定的表观易位需要过量的cidosol和阴离子两亲物的存在，GTP 7S和diC 8均增强，并且高盐抑制，定性地与活化相关;在活化剂的组合下观察到高达70%的共沉降（与不存在活化剂时< 20%相比）。使用热灭活的胞质溶胶获得了类似的结果，其中另一种氧化酶组分p67-phox已被灭活。出乎意料的是，从50%到80%的表观易位发生在膜的情况下，表明蛋白质聚集占观察到的易位的重要部分。然而，在所有情况下，由于膜的存在，易位百分比增加，表明一定程度的蛋白质-膜相互作用。虽然控制在体外翻译的蛋白未能易位，共沉淀的p47-phox发生同样好的红细胞或中性粒细胞质膜缺乏细胞色素b55 i时，使用。此外，包含在细胞色素i558大亚基的C-末端内的肽RGVHFIF不能抑制p47-phox的易位/聚集，尽管其能够抑制氧化酶的无细胞活化。SDS、diC 8和GTP 7S均作用于胞质组分以改变蛋白质-蛋白质和/或蛋白质-膜缔合，并且这些改变是必要的（B）这些改变的结合可能通过增加质膜上p47-phox和其它成分的局部浓度而起作用;（c）在无细胞激活系统中非特异性关联的高背景可能掩盖任何特异性的、功能相关的关联（例如，与细胞色素i558）;和（d）在无细胞系统中的易位机制不同于在完整的中性粒细胞中看到的。中性粒细胞[多形核白细胞（PMN）] 1是宿主抵抗微生物入侵的主要防御系统，在多种炎症状态中发挥病理作用（Rotrosen & Gallin，1987; Malech & Gallin，1987）。其抗微生物机制之一涉及呼吸爆发的激活，其利用分子氧产生超氧化物;后者其次产生其他细胞毒性物质，包括H2 O2、HOCl和羟基自由基。呼吸爆发在对抗传染病中的关键重要性由遗传性病症慢性肉芽肿病（CGD）说明，其中嗜中性粒细胞不能产生氧化剂，并且受折磨的个体遭受频繁和严重的感染（Smith& Curnutte，
Revised Manuscript Received December 20, 1991 abstract: We reported previously that diacylglycerol (diC8) and GTP7S synergize with an anionic amphiphile such as sodium dodecyl sulfate (SDS) to produce high rates of superoxide generation in a cell-free system consisting of neutrophil plasma membrane plus cytosol [Burnham, D. N., Uhlinger, D. J., & Lambeth, J. D.(1990) J. Biol. Chem. 265, 17550-17559]. Here we investigate the effects of these activating factors on the plasma membrane association in an in vitro translated radiolabeled recombinant p47-phox protein. Apparent translocation, assayed by cosedimentation with plasma membranes, required the presence of excess cjdosol and an anionic amphiphile, was enhanced by both GTP7S and diC8, and was inhibited by high salt, correlating qualitatively with activation; up to 70% cosedimentation was observed with the combination of activators (compared with< 20% in their absence). Similar results were obtained using heat-inactivated cytosol, wherein another oxidase component, p67-phox, has been inactivated. Unexpectedly, from 50 to 80% of the apparent translocation occurred in the absence of membranes, indicating that protein aggregation accounted for a significant part of the observed translocation. Nevertheless, the percent translocation was increased in all cases by the presence of membranes, indicating some degree of protein-membrane interaction. While a control in vitro translated protein failed to translocate, cosedimentation of p47-phox occurred equally well when red blood cell or neutrophil plasma membranes lacking cytochrome b55i were used. Also, the peptide RGVHFIF, which is contained within the C-terminus of the large subunit of cytochrome i558, failed to inhibit translocation/aggregation of p47-phox, despite its ability to inhibit cell-free activation of the oxidase. The data are consistent with the following:(a) SDS, diC8, and GTP7S all act on cytosolic components to alterprotein-protein and/or protein-membrane associations, and these changes are necessary (but not sufficient) for activation;(b) these altered asociations are likely to function by increasing the local con-centration of p47-phox and other components at the plasma membrane;(c) a high background of nonspecific associations in the cell-free activation system is likely to obscure any specific, functionally relevant associations (eg, with cytochrome i558); and (d) the mechanism of translocation in the cell-free system differs from that seen in intact neutrophils. e neutrophil [polymorphonuclear leukocyte (PMN)] 1 provides the primary host defense against invadingmicroor-ganisms, and can play a pathological role in a variety of inflammatory conditions (Rotrosen & Gallin, 1987; Malech & Gallin, 1987). One of its antimicrobial mechanisms involves the activation of the respiratory burst, which utilizes molecular oxygen to generate superoxide; the latter secondarily produces other cytotoxic species including H202, HOC1, and hydroxyl radical. The pivotal importance of the respiratory burst in combating infectious disease is illustrated by the inherited condition chronic granulomatous disease (CGD) wherein the neutrophils fail to generate oxidants, and afflicted individuals suffer frequent and severe infections (Smith& Curnutte,
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HL-60 粒细胞中趋化肽激活磷脂酶 D。
DOI：
10.1016/0006-291x(88)90528-1
发表时间：
1988
影响因子：
3.1
作者：
J. Pai;M. Siegel;R. Egan;M. Billah
通讯作者：
M. Billah
调节无细胞 NADPH 氧化酶激活的中性粒细胞 GTP 结合蛋白位于胞质部分。
影响因子：
4.4
作者：
T. Gabig;E. Eklund;G. B. Potter;J. Dykes
通讯作者：
J. Dykes
影响因子：
56.9
作者：
LETO, TL;LOMAX, KJ;MALECH, HL
通讯作者：
MALECH, HL
影响因子：
4.1
作者：
CROSS, AR;PARKINSON, JF;JONES, OTG
通讯作者：
JONES, OTG
1,2-二辛酰基-sn-甘油可以通过不同的机制刺激中性粒细胞。
DOI：
--
发表时间：
1989
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Badwey,JA;Robinson,JM;Heyworth,PG;Curnutte,JT
通讯作者：
Curnutte,JT