A Rise in Ionized Calcium Activates the Neutrophil NADPH-Oxidase But Is Not Sufficient to Directly Translocate Cytosolic p47phox or p67phox to b Cytochrome Containing Membranes

Neutrophil production of reactive oxygen species is dependent on an assembly process that involves a translocation of the cytosolic NADPH-oxidase components (p47_phox; p67_phox; Rac2) to a b cytochrome containing membrane. Based on the fact that an intracellular Ca²⁺ rise can activate the oxidase without any extracellular release of reactive oxygen species, we suggest that the oxidase can be assembled in a membrane distinct from the plasma membrane. Disintegrated cells were used to monitor Ca²⁺ dependent membrane binding of neutrophil cytosolic proteins. Membranes containing the b cytochrome part of the oxidase, i.e., specific granules and plasma membranes/secretory vesicles, were used in the translocation experiments. Several cytosolic proteins were found to translocate to specific granules as well as the plasma membranes/secretory vesicles, one of them being annexin I. Using antibodies in the blotting assay against the cytosolic oxidase components p47_phox and p67_phox, we could show that no Ca²⁺ dependent translocation of these cytosolic proteins occur to neither of the b cytochrome containing membranes.