Biochemical properties of the NADPH oxidase in neutrophils

Kakinuma, Katsuyoshi; Yamaguchi, Teruhide; Fukuhara, Yukiko; Kaneda, Mizuho; Chiba, Takamitsu

doi:10.2492/jsir1981.4.4_413

Cited by 5 publications

(16 citation statements)

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“…The flavin enzyme was separated by means of disc IEF-PAGE (5.0 x 60 ram; diameter x length) as in [1,14]. IEF was carried out for a total of 1700 V-h in a refrigerator at 2°C.…”

Section: Iefmentioning

confidence: 99%

“…Stimulated and resting neutrophils were disrupted by sonication to isolated membrane vesicles as reported in [1].…”

Section: 2 Neutrophils and Membrane Vesiclesmentioning

confidence: 99%

“…Membrane-bound NADPH oxidase was solubilized using 0.75070 octyl glucoside, 3007o glycerol and 0.19 M sucrose, in 50 mM K-Na phosphate buffer, pH 7.4 [1], in the presence or absence of the following protease inhibitors: lenpeptin (50 /zg/ml), chymostatin (50/zg/ml), pepstatin (50/zg/ml), PMSF (1 mM) and EGTA (0.1 raM) at 0°C for 30 min. Each mixture was centrifuged at 105 x g for 60 min in a Beckman 60 Ti rotor.…”

Section: Solubilization Of Nadph Oxidasementioning

confidence: 99%

“…Abbreviations: IEF-PAGE, isoelectric focusing-polyacrylamide gel electrophoresis; SDS-PAGE, SDS-polyacrylamide gel electrophoresis; NBT, nitroblue tetrazolium zyme, which is possibly part of the respiratory burst oxidase, was separated by IEF-PAGE in our laboratory [1]. The enzyme, which focused at pI 5.0, showed characteristics similar to those of the oxidase and a high specificity for NADPH.…”

Section: Introductionmentioning

confidence: 99%

“…This activity also appears when neutrophils are stimulated with membraneperturbating agents [6,7]. While the nature of the NADPH oxidase has not yet been elucidated, recent studies have shown that it may consist of multiple components, a b-type cytochrome [8,9] and a flavoprotein [1,10]. Recently, an FAD enCorrespondence address: Y. Fukuhara, Tokyo Metropolitan Institute of Medir.al Science, 18-22, Honkomagome 3-chome, Bunkyo-ku, Tokyo 113, Japan…”

Section: Introductionmentioning

confidence: 99%

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Immunological studies on the respiratory burst oxidase of pig blood neutrophils

1988

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Recently, a flavin enzyme (pI 5.0), that is probably responsible for superoxide (O~)-generating oxidase activity, was separated by isoelectric focusing-polyacrylamide gel electrophoresis (IEF-PAGE) from neutrophil membranes in our laboratory [(1987) J. Biol. Chem. 262, 12316-12322]. In the present work, we performed immunological studies on this enzyme derived from pig blood neutrophils. The enzyme extract obtained on IEF-PAGE was injected into guinea pigs to raise antibodies. IgG antibody against the pI 5.0 protein inhibited maximally 54% of the O~--generating activity of the membrane-solubilized oxidase, whereas the normal serum IgG was not inhibitory at all. Our results further confirmed that the enzyme (pl 5.0) is one of the component(s) of the O;-generating system. The enzyme gave rise to a band corresponding to a major protein of 72_ 4 kDa on both non-denaturing and SDS-PAGE. Immunoblotting after SDS-PAGE demonstrated labelling of peptides of 70-72, 28-32 and 16-18 kDa.

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“…The flavin enzyme was separated by means of disc IEF-PAGE (5.0 x 60 ram; diameter x length) as in [1,14]. IEF was carried out for a total of 1700 V-h in a refrigerator at 2°C.…”

Section: Iefmentioning

confidence: 99%

“…Stimulated and resting neutrophils were disrupted by sonication to isolated membrane vesicles as reported in [1].…”

Section: 2 Neutrophils and Membrane Vesiclesmentioning

confidence: 99%

Section: Solubilization Of Nadph Oxidasementioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Immunological studies on the respiratory burst oxidase of pig blood neutrophils

1988

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The Respiratory Burst Oxidase

Babior

1992

Advances in Enzymology - And Related Areas of Molecular Biology

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Nitric Oxide Inactivates NADPH Oxidase in Pig Neutrophils by Inhibiting Its Assembling Process

Fujii

Ichimori

Hoshiai

et al. 1997

Journal of Biological Chemistry

137

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Nitric oxide (NO) is now recognized as one of the key mediators in many physiological and pathological processes (see reviews in Refs. 1 and 2). NO is also known to be a multifunctional molecule, one function of which is to inactivate biologically important enzymes such as mitochondrial respiratory enzymes and GAPDH, which play important roles in energy production (3, 4), ribonucleotide reductase, which is the key enzyme for protein synthesis (5), and the superoxide-generating enzymes, NADPH oxidase (6) and xanthine oxidase (7). Particularly important is the effect of NO on NADPH oxidase, because under conditions such as inflammation, the accumulation of phagocytes is a common feature and the induction of NO synthase has been shown. It is plausible that increased formation of NO interferes with the activity of NADPH oxidase and reduces superoxide (O 2 . ) production (8, 9). Despite the importance of the NO effect on NADPH oxidase, no detailed study has been carried out since the initial report by Clancy et al. (6) in which inhibition of O 2 . generation by NO was demonstrated. The underlying mechanism was suggested to be the direct interaction of NO on the membrane components of NADPH oxidase (6). The NADPH oxidase of phagocytes is a multi-component electron transport system, in which activation requires the assembly of three cytosolic regulatory proteins (p47 phox , p67 phox , and Rac1/Rac2) to membrane-bound cytochrome b 558 (10, 11). Cytochrome b 558 is postulated to be a membranebound flavocytochrome with six-coordinated low spin heme and FAD as redox centers. The electrons provided by NADPH are thought to be transferred in a linear sequence, NADPH 3 FAD 3 heme (Fe 3ϩ ) 3 O 2 . The heme in cytochrome b 558 is assumed to be the terminal electron donor in the production of O 2. from molecular oxygen due to its unusually low redox potential, Ϫ245 mV (12). Although the most plausible site of NADPH oxidase attacked by NO was suggested to be in membrane protein(s) (6), a detailed analysis of these effects has not been performed. Considering that nitrosyl-iron complex easily forms in heme-containing enzymes (3), the heme structure of cytochrome b 558 and the electron flux from substrate (NADPH) to redox centers, FAD and low spin heme, in NADPH oxidase should be examined to clarify the effects of NO on its O 2 . -generating activity.In the present study, we examined the effects of NO on electron fluxes in neutrophil NADPH oxidase. Under aerobic conditions the effects of NO on O 2 . -generating activity of NADPH oxidase (reaction 1) was examined by the cytochrome c reduction method. In this study, we also employed the solubilized NADPH oxidase obtained from stimulated cells and measured its O 2 . -generating activity in the presence of NO.Under anaerobic conditions the effects of NO on the electron transfer reaction in each redox center was examined: NADPH 3 FAD 3 exogenous electron acceptor, cytochrome c (reaction 2) and NADPH 3 FAD 3 cytochrome b 558 (reaction 3). Under both aerobic and anaerobic conditions, the...

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Biochemical properties of the NADPH oxidase in neutrophils

Cited by 5 publications

References 0 publications

Immunological studies on the respiratory burst oxidase of pig blood neutrophils

Immunological studies on the respiratory burst oxidase of pig blood neutrophils

The Respiratory Burst Oxidase

Nitric Oxide Inactivates NADPH Oxidase in Pig Neutrophils by Inhibiting Its Assembling Process

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