2001
DOI: 10.1074/jbc.m007759200
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Phosphatidic Acid and Diacylglycerol Directly Activate NADPH Oxidase by Interacting with Enzyme Components

Abstract: The enzyme NADPH oxidase is regulated by phospholipase D in intact neutrophils and is activated by phosphatidic acid (PA) plus diacylglycerol (DG) in cell-free systems. We showed previously that cell-free NADPH oxidase activation by these lipids involves both protein kinase-dependent and -independent pathways. Here we demonstrate that only the protein kinase-independent pathway is operative in a cell-free system of purified and recombinant NADPH oxidase components. Activation by PA ؉ DG was ATP-independent and… Show more

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Cited by 112 publications
(79 citation statements)
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References 63 publications
(78 reference statements)
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“…Such effects were postulated to mimic in vivo phosphorylation events where Src homology 3 domain interactions are believed to partly control oxidase activity. Other experiments conducted by our lab (26,27) and others provide evidence that the amphiphiles also interact directly with Cyt b to impart similar conformational effects that may effect partial oxidase control. Collectively, these studies suggest that that the amphiphiles interact with several components of the oxidase to induce full activity.…”
Section: Translocation Assaymentioning
confidence: 99%
“…Such effects were postulated to mimic in vivo phosphorylation events where Src homology 3 domain interactions are believed to partly control oxidase activity. Other experiments conducted by our lab (26,27) and others provide evidence that the amphiphiles also interact directly with Cyt b to impart similar conformational effects that may effect partial oxidase control. Collectively, these studies suggest that that the amphiphiles interact with several components of the oxidase to induce full activity.…”
Section: Translocation Assaymentioning
confidence: 99%
“…Separation of Membrane and Cytosolic Fractions-Membrane and cytosolic fractions of NOX5 and control-transfected HEK293 cells were prepared as described for neutrophil granulocytes (33). Briefly, cells were resuspended in sonication buffer (11% sucrose, 0.2ϫ PBS, pH 7.5, 120 mM NaCl, 5 mM EGTA, 1 mM PMSF) and broken by sonication.…”
Section: Methodsmentioning
confidence: 99%
“…Sonicates were centrifuged (200 ϫ g, 10 min) and the supernatant was layered onto a 17/40% (w/v) discontinuous sucrose gradient and centrifuged (150,000 ϫ g for 30 min). Cytosolic fractions were collected from the top layer, and membrane fractions were collected from the 17/40% interface (33 mM KCl, containing 1 mM EGTA) and put on a 0.8 ϫ 40 cm Sephadex G-25 (for NOX5-EF) or P10 column (for C-NOX5-EF and N-NOX5-EF) equilibrated in buffer A. The protein concentration was measured spectrophotometrically using molar extinction coefficients at 278 nm of 18,100, 11,000, and 7100 M Ϫ1 cm Ϫ1 for NOX5-EF (and E31Q mutant), N-NOX5-EF, and C-NOX5-EF, respectively.…”
Section: Methodsmentioning
confidence: 99%
“…generation Membranes of NOX5-tranfected and control HEK293 cells were prepared as described before [15,20]. Superoxide generation was measured by lucigenin chemiluminescence assay using FluoSTAR OPTI-MA, BMG Labtech.…”
Section: Membrane Preparation and Detection Of Nadph Superoxidementioning
confidence: 99%