Relationships of p40(phox) with p67(phox) in the activation and expression of the human respiratory burst NADPH oxidase.
p40(phox) of the phagocyte NADPH oxidase forms a complex with p67(phox) in cytosol, and coincidentally decreases in patients who lack p67(phox). Here we investigated the mode of translocation of p40(phox) to the membrane, its cytoskeletal localization on activation of the NADPH oxidase, and the dependency of its expression relative to that of p67(phox). When human polymorphonuclear leukocytes (PMNs) were stimulated with phorbol myristate acetate (PMA), p40(phox) was translocated to the membrane along with p67(phox), and not was released into the cytosol. Studies with resting PMNs using Triton X-100 revealed the exclusive localization of p67(phox) in the cytoskeletal fraction. Unexpectedly, however, about half of p40(phox), which is deemed to be fully associated with p67(phox), was recovered in the non-cytoskeletal fraction. Unlike p47(phox), the association of p40(phox) with cytoskeleton was not induced by the PMA-stimulation. These results indicate not only that p40(phox) associates with cytoskeleton via a molecule of p67(phox), but also that there are distinct states of p40(phox) that can be manipulated with Triton X-100. Lastly, Western-blot analysis of hematopoietic cells revealed no correlation between p40(phox) and p67(phox) in their protein expressions during cell differentiation, and also that p40(phox) can be stably present alone in cells, unless in the case of mature PMNs. In this regard, definitive proof was obtained with Epstein-Barr virus-transformed B cells of a p67(phox)-deficient patient, in which p40(phox) was normally expressed.
Tsunawaki
S
S
Department of Infectious Disease, National Children's Medical Research Center, Taishido, Setagaya-ku, Tokyo 154-8509, Japan. tsunawaki-s@nch.go.jp
Yoshikawa
K
K
D015536:Down-Regulation; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D015971:Gene Expression Regulation, Enzymologic; D018922:HL-60 Cells; D006801:Humans; D009247:NADH, NADPH Oxidoreductases / Q000096:biosynthesis / Q000378:metabolism*; D019255:NADPH Oxidases; D009504:Neutrophils / Q000187:drug effects; D010750:Phosphoproteins / Q000494:pharmacology*; D016176:T-Lymphocyte Subsets / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D010750:Phosphoproteins; C085072:neutrophil cytosol factor 40K; C079789:neutrophil cytosol factor 67K; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases; C022709:superoxide-forming enzyme; D013755:Tetradecanoylphorbol Acetate