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标题：Properties of NADPH Oxidase in Specific Granule-Rich Fraction Prepared from Guinea Pig Neutrophils
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作者：Naoko ABE ; Naoki OKAMURA ; Sadahiko ISHIBASHI 等
期刊名称：Biological and Pharmaceutical Bulletin
印刷版ISSN：0918-6158
电子版ISSN：1347-5215
出版年度：2000
卷号：23
期号：5
页码：537-541
DOI：10.1248/bpb.23.537
出版社：The Pharmaceutical Society of Japan
摘要：Both the plasma membrane-rich fraction and specific granule-rich fraction prepared from human neutrophil lysate by Percoll centrifugation have been reported to contain cytochrome b₅₅₈, a membrane activation factor for NADPH oxidase. In this study, the plasma membrane-rich fraction and specific granule-rich fraction of guinea pig neutrophils were prepared, and the abilities of both fractions to activate NADPH oxidase in a cellfree system consisting of either fraction, cytosol and arachidonate were compared. There was no difference in the K_m value for NADPH between NADPH oxidase activated by specific granules or by plasma membranes. Optimum concentrations of arachidonate for the activation of NADPH oxidase in both the fractions were also the same. However, after freeze-thawing, the specific granules markedly lost the ability, compared to plasma membranes. Such instability of specific granules was also observed on hypotonic- or deoxycholate-treatment. The inactivation by freeze-thawing was not suppressed by proteinase inhibitors, and gp91-phox, a large subunit of cytochrome b₅₅₈, was not degraded by freeze-thawing. Freeze-thawed specific granules did not affect the ability in plasma membranes, indicating the absence of an inactivating factor in specific granules. The increase in the amount of cytosol in the cell-free assay mixture did not compensate for the markedly decreased ability of freezethawed specific granules. Translocation of p47-phox, one of the cytosolic activation factors, to specific granules was not affected by freeze-thawing. We found that the ability of specific granules to activate NADPH oxidase was fragile, though it is unclear what is responsible for the instability, at present.
关键词：NADPH oxidase;guinea pig;specific granule;freeze-thaw;cell-free activation