A neutrophil GTP-binding protein that regulates cell free NADPH oxidase activation is located in the cytosolic fraction.
A neutrophil GTP-binding protein that regulates cell free NADPH oxidase activation is located in the cytosolic fraction.
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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。
DOI：
10.4049/jimmunol.145.3.945
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发表时间：
1990
影响因子：
4.4
通讯作者：
J. Dykes
中科院分区：
文献类型：
--
作者：
T. Gabig;E. Eklund;G. B. Potter;J. Dykes
在花生四烯酸酯和多组分细胞质组分的存在下，未经刺激的中性粒细胞在质膜中休眠的产生氧（-）的氧化酶被激活。这个过程是由不可水解的GTP类似物刺激的，可能涉及百日咳毒素不敏感的GTP结合蛋白。我们的研究旨在表征假定的gtp结合蛋白，将其定位于该系统的膜或细胞质部分。将分离的膜组分暴露于鸟苷-5'-（3- o -硫）三磷酸（GTP γ S）中，无论是否含有花生四烯酸酯，对细胞质组分随后的NADPH氧化酶活化没有影响。单独将细胞质部分预先暴露于GTP γ S并不能增强膜氧化酶的激活。然而，将细胞质预先暴露于GTP γ - S然后花生四烯酸导致其激活膜氧化酶的能力增强四倍。在去除未结合的GTP γ S和花生四烯酸酯后，这种增强是明显的，并且在膜激活过程中，额外的GTP γ S不会增强这种增强。重建分析开发了细胞质成分(s)负责GTP γ s效应。用GTP - 35S预孵育细胞质溶胶，用阴离子交换色谱法分离花生四烯酸酯。蛋白结合的GTP γ 35S单峰被恢复，具有重构活性。与GTP γ 35S单独预孵育的细胞质溶胶进行类似的分离，观察到相同的蛋白结合GTP γ 35S峰。然而，该峰不具有重构活性。我们得出结论，gtp结合蛋白调节这个无细胞系统位于细胞质部分。该蛋白的GTP γ s配体形式可能被花生四烯酸激活或稳定。
The dormant O2(-)-generating oxidase in plasma membranes from unstimulated neutrophils becomes activated in the presence of arachidonate and a multicomponent cytosolic fraction. This process is stimulated by nonhydrolyzable GTP analogues and may involve a pertussis toxin insensitive GTP-binding protein. Our studies were designed to characterize the putative GTP-binding protein, localizing it to either membrane or cytosolic fraction in this system. Exposure of the isolated membrane fraction to guanosine-5'-(3-O-thio)triphosphate (GTP gamma S), with or without arachidonate, had no effect on subsequent NADPH oxidase activation by the cytosolic fraction. Preexposure of the cytosolic fraction to GTP gamma S alone did not enhance activation of the membrane oxidase. However, preexposure of the cytosol to GTP gamma S then arachidonate caused a four-fold enhancement of its ability to activate the membrane oxidase. This enhancement was evident after removal of unbound GTP gamma S and arachidonate, and was not augmented by additional GTP gamma S during membrane activation. A reconstitution assay was developed for cytosolic component(s) responsible for the GTP gamma S effect. Cytosol preincubated with GTP gamma 35S then arachidonate was fractionated by anion exchange chromatography. A single peak of protein-bound GTP gamma 35S was recovered that had reconstitutive activity. Cytosol preincubated with GTP gamma 35S alone was similarly fractionated and the same peak of protein-bound GTP gamma 35S was observed. However, this peak had no reconstitutive activity. We conclude that the GTP-binding protein regulating this cellfree system is located in the cytosolic fraction. The GTP gamma S-liganded form of this protein may be activated or stabilized by arachidonate.