Properties of NADPH oxidase in specific granule-rich fraction prepared from guinea pig neutrophils.
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 cell-free system consisting of either fraction, cytosol and arachidonate were compared. There was no difference in the Km 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 freeze-thawed 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.
Abe
N
N
Department of Physiological Chemistry, Faculty of Medicine, Hiroshima University, Japan.
Okamura
N
N
Ishibashi
S
S
D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D002474:Cell-Free System; D003594:Cytoplasmic Granules / Q000378:metabolism; D010450:Endopeptidases / Q000378:metabolism; D004789:Enzyme Activation; D005260:Female; D006168:Guinea Pigs; D006801:Humans; D007700:Kinetics; D019255:NADPH Oxidases / Q000378:metabolism*; D009504:Neutrophils / Q000166:cytology / Q000201:enzymology*; D013347:Subcellular Fractions / Q000201:enzymology
D019255:NADPH Oxidases; D010450:Endopeptidases