Activation of a NADPH Oxidase From Horse Polymorphonuclear Leukocytes in a Cell- Free System

Heyneman, Roger; Vercauteren, R.

doi:10.1002/jlb.36.6.751

Cited by 212 publications

(58 citation statements)

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“…These include the activation of protein kinases to phosphorylate cellular proteins and NADPH oxidase components (2,8) and the generation of various lipid second messengers (AA by phospholipase A 2 (9); DG by phospholipase C or PA phosphohydrolase; PA by phospholipase D or DG kinase (10,11)). In cell-free systems, these lipids can induce activation of the enzyme (12)(13)(14)(15)(16)(17). AA exerts its effect by directly acting on enzyme components (18 -22).…”

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confidence: 99%

Phosphatidic Acid and Diacylglycerol Directly Activate NADPH Oxidase by Interacting with Enzyme Components

Palicz

Foubert

Jesaitis

et al. 2001

Journal of Biological Chemistry

112

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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 unaffected by the protein kinase inhibitor staurosporine, indicating the lack of protein kinase involvement. Both PA and DG were required for optimal activation to occur. The drug R59949 reduced activation of NADPH oxidase by either arachidonic acid or PA ؉ DG, with IC 50 values of 46 and 25 M, respectively. The optimal concentration of arachidonic acid or PA ؉ DG for oxidase activation was shifted to the right with R59949, indicating interference of the drug with the interaction of lipid activators and enzyme components. R59949 inhibited the lipid-induced aggregation/sedimentation of oxidase components p47 phox and p67 phox , suggesting a disruption of the lipidmediated assembly process. The direct effects of R59949 on NADPH oxidase activation complicate its use as a "specific" inhibitor of DG kinase. We conclude that the protein kinase-independent pathway of NADPH oxidase activation by PA and DG involves direct interaction with NADPH oxidase components. Thus, NADPH oxidase proteins are functional targets for these lipid messengers in the neutrophil.The NADPH oxidase (the respiratory burst enzyme) in phagocytic cells produces superoxide (O 2 . ) 1 by catalyzing electron transfer from NADPH to molecular oxygen upon cell stimulation (1-3). This enzyme plays important roles in host defense against infection and in tissue damage due to inflammation (1-5). In addition, NADPH oxidase-like enzymes are present in a variety of other cell types, where the oxygen radicals formed may have signaling roles (5, 6). The enzyme in phagocytes consists of the membrane-bound heterodimeric flavocytochrome b 558 (gp91 phox and p22 phox ) and four cytosolic proteins (p47 phox , p67 phox , p40 phox , Rac1/2) (2-4, 7). Components must assemble in the membrane for the enzyme to become active (2-4, 7). The activation of NADPH oxidase is initiated by receptor-ligand interaction and involves complex intracellular signaling events. These include the activation of protein kinases to phosphorylate cellular proteins and NADPH oxidase components (2, 8) and the generation of various lipid second messengers (AA by phospholipase A 2 (9); DG by phospholipase C or PA phosphohydrolase; PA by phospholipase D or DG kinase (10, 11)). In cell-free systems, these lipids can induce activation of the enzyme (12-17). AA exerts its effect by directly acting on enzyme components (18 -22). PA has been shown to partially activate purified flavocytochrome b 558 (23), suggesting it interacts with this protein. It is not known whether DG has any direct effe...

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confidence: 99%

Phosphatidic Acid and Diacylglycerol Directly Activate NADPH Oxidase by Interacting with Enzyme Components

Palicz

Foubert

Jesaitis

et al. 2001

Journal of Biological Chemistry

112

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“…Rossi, 1986;Bellavite, 1988;Segal, 1989). It is thought that in activated neutrophils the NADPH oxidase complex is composed of two redox components, an NADPHdependent dehydrogenase of molecular mass 65 kDa (Doussi2re and Vignais, 1985;Markert et al, 1985;Kakinuma et al, 1987), that probably contains FAD as prosthetic group, and a low-potential cytochrome b referred to as cytochrome 6 5 5 8 because of the position of its tl band of light absorbance (for review, see Segal, 1989). In a recent paper, it was postu- lated that activation of the respiratory-burst oxidase could be due to the translocation of an NADPH-binding protein from cytosol to the plasma membrane of the cell (Smith et al, 1989).…”

Section: Respiratory Burst Of Rabbit Peritoneal Neutrophilsmentioning

confidence: 99%

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Respiratory burst of rabbit peritoneal neutrophils

Laporte

Doussière

Vignais

1990

European Journal of Biochemistry

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Superoxide COY) production by the NADPH oxidase of a membrane fraction derived from rabbit peritoneal neutrophils activated by 4P-phorbol1Zmyristate 13-acetate (PMA) was studied at 25 "C under different conditions, and measured by the superoxide dismutase inhibitable reduction of cytochrome c. Whereas PMA-activated rabbit neutrophils incubated in a glucose-supplemented medium exhibited a substantial rate of production of ' O;, the membranes prepared by sonication of the activated neutrophils were virtually unable to generate '0, in the presence of NADPH. Instead, they exhibited an NADPH-dependent diaphorase activity, measured by the superoxide-dismutase-insensitive reduction of cytochrome c. Upon addition of arachidonic acid, which is known to elicit oxidase activation, the NADPH diaphorase activity of the rabbit neutrophil membranes vanished and was stoichiometrically replaced by an NADPH oxidase activity. The emerging oxidase activity was fully sensitive to iodonium biphenyl, a potent inhibitor of the respiratory burst, whereas the diaphorase activity was not affected. Addition of 0.1 % Triton X-100 or an excess of arachidonic acid, acting as detergent, resulted in the reappearance of the diaphorase activity at the expense of the oxidase activity. These results indicate that the diaphoraseoxidase transition is reversible. When the rabbit neutrophil membranes were supplemented with rabbit neutrophil cytosol, guanosine 5'-[pthio]triphosphate and Mg2+, in addition to arachidonic acid, not only the NADPH diaphorase activity disappeared, but the emerging NADPH oxidase activity was markedly enhanced (about 10 times compared to that of membranes treated with arachidonic acid alone). The diaphorase -oxidase transition was accompanied by a 10-fold increase in the K , for NADPH, suggesting a change of conformation propagated to the NADPH-binding site during the transition. The treatment of PMA-activated rabbit neutrophils with crosslinking reagents, like glutaraldehyde or l-(3-dimethylaminopropyl)-3-ethyl carbodiimide, prevented the loss of the PMA-elicited oxidase activity upon disruption of the cells by sonication, suggesting that the interactions between the components of the oxidase complex are stabilized by cross-linking.Phagocytes exposed to a variety of soluble and particulate stimuli show a rapid production of the superoxide anion 'O;, by reduction of 02. The enzyme responsible is an NADPH oxidase complex. This reaction is known as the respiratory burst, since the oxidase is dormant in resting phagocytes and is powerfully activated upon the binding of stimulatory ligands on specific membrane receptors (for review, cf. Rossi, 1986; Bellavite, 1988; Segal, 1989). It is thought that in activated neutrophils the NADPH oxidase complex is composed of two redox components, an NADPHdependent dehydrogenase of molecular mass 65 kDa (Doussi2re and Vignais, 1985;Markert et al., 1985;Kakinuma et al., 1987), that probably contains FAD as prosthetic group, and a low-potential cytochrome b referred to as cytochrome 6 5 5 8 beca...

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“…Recently, the activating process has been studied in disrupted unstimulated phagocytes [3]. The oxidase in the homogenates of neutrophils can be activated by SDS [7][8][9][10] or fatty acids such as arachidonic acid and oleic acid [3][4][5][6][8][9][10][11][12][13]. Both cytosol and membrane fractions are necessary for the activation.…”

Section: Introductionmentioning

confidence: 99%

GTP‐dependent and ‐independent activation of superoxide producing NADPH oxidase in a neutrophil cell‐free system

et al. 1989

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GTP and GTP-~,-S enhanced several-fold the NADPH-depcndent supcroxide production induced by sodium dodeeyl sulfate in a eell-free system of pig neutrophils consisting of the mebrane fraction and two cytosolic fractions separated by gel filtration. The enhanced activity was decreased by the addition of GDP in a dose-dgl~endcnt manner, but 70~ .of the activity in the absenee of GTP remained even at 1 mM GDP. Only one cytosol fraction besides the membrane fraction was required for the activation in the presence of GTP. The cytosol fraction was analyzed by chromatography on 2%5'-ADP agarose and two components responsible for the (3TP-depcndent and independent activation were separated. These results suggest that at least two pathways are available for the activation of supcroxide production in the cell-free system of pig neutrophils.

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Activation of a NADPH Oxidase From Horse Polymorphonuclear Leukocytes in a Cell- Free System

Cited by 212 publications

References 23 publications

Phosphatidic Acid and Diacylglycerol Directly Activate NADPH Oxidase by Interacting with Enzyme Components

Phosphatidic Acid and Diacylglycerol Directly Activate NADPH Oxidase by Interacting with Enzyme Components

Respiratory burst of rabbit peritoneal neutrophils

GTP‐dependent and ‐independent activation of superoxide producing NADPH oxidase in a neutrophil cell‐free system

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