Remarkable stabilization of neutrophil NADPH oxidase using RacQ61L and a p67phox-p47phox fusion protein.
Activation of the phagocyte NADPH oxidase occurs via assembly of cytosolic p47(phox), p67(phox), and Rac with the membrane-bound flavocytochrome b(558). Recently, we have found that p67(phox)-(1-210) (p67N) fused with p47(phox)-(1-286) (p47N) or with Rac efficiently stabilizes the oxidase in a cell-free reconstitution system. In an attempt to further stabilize the oxidase, we herein used a constitutively active Rac, RacQ61L, and examined its effect on the oxidase stability. The half-life (t(1/2)) of the activity reconstituted with wild-type Rac was 12 min at 37 degrees C, which was extended 6-fold by RacQ61L. Also, the stability of the oxidase without p47(phox) increased 8-fold using RacQ61L. RacQ61L had a higher affinity for the complex than wild-type Rac and increased the affinity of p67N for the complex. Far-western blotting showed an enhanced binding between RacQ61L and p67N. The oxidase was stabilized by nanomolar FAD, and RacQ61L lowered the FAD concentration required. The combination of RacQ61L and a fusion protein consisting of p67N and p47N produced an extremely stable enzyme (t(1/2) = 184 min at 37 degrees C). The effectiveness of RacQ61L and fusion proteins on stabilization was in the following order: p67N-Rac < p67N + RacQ61L < or = p67N-RacQ61L << p67N-p47N + RacQ61L. These results indicate that a tightly bound ternary complex of p67(phox), Rac, and p47(phox) is very effective in maintaining the oxidase and confirm that the longevity of the activated state requires continuous association of these components. This simple and efficient method of stabilization may provide a useful tool to elucidate the nature of the activated oxidase.
Miyano
Kei
K
Department of Applied Chemistry, Faculty of Engineering, Ehime University, Matsuyama, Ehime 790-8577, Japan.
Fukuda
Hiroyasu
H
Ebisu
Kentaro
K
Tamura
Minoru
M
D004789:Enzyme Activation / Q000235:genetics; D004795:Enzyme Stability / Q000235:genetics; D005182:Flavin-Adenine Dinucleotide / Q000737:chemistry; D020558:GTP Phosphohydrolases / Q000737:chemistry / Q000235:genetics; D005973:Glutamine / Q000235:genetics; D006150:Guanine Nucleotides / Q000737:chemistry; D006358:Hot Temperature; D006801:Humans; D007700:Kinetics; D007930:Leucine / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D019255:NADPH Oxidases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D009504:Neutrophils / Q000201:enzymology* / Q000378:metabolism; D010750:Phosphoproteins / Q000737:chemistry / Q000172:deficiency / Q000235:genetics*; D011485:Protein Binding / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000138:chemical synthesis / Q000737:chemistry*; D013816:Thermodynamics; D020744:rac GTP-Binding Proteins / Q000235:genetics*
D006150:Guanine Nucleotides; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; C079789:neutrophil cytosol factor 67K; D005973:Glutamine; D005182:Flavin-Adenine Dinucleotide; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1; D020558:GTP Phosphohydrolases; D020744:rac GTP-Binding Proteins; D007930:Leucine