Translocation of p21rac2 from cytosol to plasma membrane is neither necessary nor sufficient for neutrophil NADPH oxidase activity.
Activation of the membrane-associated NADPH oxidase of neutrophils requires several cytosolic factors including p47phox, p67phox and p21rac2. We compared NADPH oxidase activity with the membrane translocation of p47phox, p67phox, and p21rac2. In a cell-free system, GTP gamma S stimulated translocation of p47phox and p67phox to the plasma membrane only in the presence of arachidonate, and this translocation correlated with NADPH oxidase activity of the reisolated plasma membranes (R = 0.94 and 0.97, respectively). In contrast, GTP gamma S-stimulated p21rac2 translocation with or without arachidonate, and the extent of translocation did not correlate with oxidase activity (R = 0.17). Neutrophil cytoplasts were used to relate membrane translocation of p47phox, p67phox and p21rac2 to membrane oxidase activity in response to the inflammatory agonists. Whereas N-formyl-methionyl-leucyl-phenylalanine stimulated equimolar, transient membrane translocation of p47phox and p67phox which kinetically paralleled NADPH oxidase activity, relatively little p21rac2 translocated (moles of p47phox/p21rac2 = 16.6). Moreover, although phorbol 12-myristate 13-acetate stimulated both the stable translocation of p47phox and p67phox and sustained NADPH oxidase activity, it did not stimulate p21rac2 translocation. From these data we conclude that membrane translocation of p21rac2 does not regulate NADPH oxidase activity stoichiometrically.
Philips
M R
MR
Department of Medicine, New York University School of Medicine, New York 10016, USA.
Feoktistov
A
A
Pillinger
M H
MH
Abramson
S B
SB
D002458:Cell Fractionation; D002462:Cell Membrane / Q000378:metabolism; D002474:Cell-Free System; D003600:Cytosol / Q000378:metabolism; D020558:GTP Phosphohydrolases / Q000097:blood*; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006150:Guanine Nucleotides / Q000494:pharmacology*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009247:NADH, NADPH Oxidoreductases / Q000097:blood*; D019255:NADPH Oxidases; D009504:Neutrophils / Q000201:enzymology*; D010750:Phosphoproteins / Q000097:blood; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D020744:rac GTP-Binding Proteins
D006150:Guanine Nucleotides; D010750:Phosphoproteins; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D009240:N-Formylmethionine Leucyl-Phenylalanine; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D020744:rac GTP-Binding Proteins; D013755:Tetradecanoylphorbol Acetate