Separation of human neutrophil plasma membrane from intracellular vesicles containing alkaline phosphatase and NADPH oxidase activity by free flow electrophoresis.

Abstract Phorbol esters are potent stimulants of the respiratory burst of the human neutrophil as assessed by superoxide (O2-) generation in whole cells and by NADPH-oxidase activity in a broken-cell 27,000-g particulate fraction. Phorbol 12-myristate, 13-acetate (PMA) and phorbol 12,13-dibutyrate (PDBu) stimulate production of O2- by human neutrophils with ED50 concentrations of 3.9 +/- 2.1 and 41.7 +/- 7.1 nM, respectively. The relation of biologic activity to receptor occupancy was assessed with binding studies of PMA and PDBu. Phorbol ester binding revealed a single high affinity phorbol ester receptor present at 7.6 x 10(5) sites/cell. The binding affinities for PMA and PDBu, 4.9 nM and 38.4 nM, respectively, agreed quantitatively with that of biologic potencies. Because of the high concentration of phorbol ester receptors (up to 125 nM) and the large amount of nonspecific binding at high cell density, apparent discrepancies between ED50′s for NADPH-oxidase and whole cell O2- generation were noted. With the use of low cell concentrations, quantitative agreement between intact cell production of O2-, NADPH-oxidase activity, and receptor binding was found. These results further support the identity of the NADPH-oxidase as the enzymatic source of respiratory burst O2- production in human neutrophils.

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Priming by tumor necrosis factor-α of human neutrophil NADPH-oxidase activity induced by anti-proteinase-3 or anti-myeloperoxidase antibodies

Journal of Leukocyte Biology ◽

10.1189/jlb.0304144 ◽

2006 ◽

Vol 80 (6) ◽

pp. 1424-1433 ◽

Cited By ~ 15

Author(s):

Dominique Reumaux ◽

Peter L. Hordijk ◽

Patrick Duthilleul ◽

Dirk Roos

Keyword(s):

Tumor Necrosis Factor ◽

Nadph Oxidase ◽

Human Neutrophil ◽

Oxidase Activity ◽

Tumor Necrosis ◽

Tumor Necrosis Factor Α ◽

Proteinase 3 ◽

Nadph Oxidase Activity ◽

Factor Α ◽

Necrosis Factor

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Remodeling of the plasma membrane after stimulation of neutrophils with f-Met-Leu-Phe and dihydrocytochalasin B: identification of membrane subdomains containing NADPH oxidase activity

Journal of Leukocyte Biology ◽

10.1002/jlb.55.6.685 ◽

1994 ◽

Vol 55 (6) ◽

pp. 685-694 ◽

Cited By ~ 24

Author(s):

Goutam Mukherjee ◽

Mark T. Quinn ◽

John G. Linner ◽

Algirdas J. Jesaitis

Keyword(s):

Plasma Membrane ◽

Nadph Oxidase ◽

Oxidase Activity ◽

Nadph Oxidase Activity ◽

Stimulation Of

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Relation of human neutrophil phorbol ester receptor occupancy and NADPH- oxidase activity

Blood ◽

10.1182/blood.v60.2.333.bloodjournal602333 ◽

1982 ◽

Vol 60 (2) ◽

pp. 333-339

Author(s):

AI Tauber ◽

DB Brettler ◽

EA Kennington ◽

PM Blumberg

Keyword(s):

Nadph Oxidase ◽

Phorbol Ester ◽

Respiratory Burst ◽

Human Neutrophil ◽

Oxidase Activity ◽

Receptor Occupancy ◽

Human Neutrophils ◽

Binding Studies ◽

Nadph Oxidase Activity ◽

Phorbol Ester Receptor

Phorbol esters are potent stimulants of the respiratory burst of the human neutrophil as assessed by superoxide (O2-) generation in whole cells and by NADPH-oxidase activity in a broken-cell 27,000-g particulate fraction. Phorbol 12-myristate, 13-acetate (PMA) and phorbol 12,13-dibutyrate (PDBu) stimulate production of O2- by human neutrophils with ED50 concentrations of 3.9 +/- 2.1 and 41.7 +/- 7.1 nM, respectively. The relation of biologic activity to receptor occupancy was assessed with binding studies of PMA and PDBu. Phorbol ester binding revealed a single high affinity phorbol ester receptor present at 7.6 x 10(5) sites/cell. The binding affinities for PMA and PDBu, 4.9 nM and 38.4 nM, respectively, agreed quantitatively with that of biologic potencies. Because of the high concentration of phorbol ester receptors (up to 125 nM) and the large amount of nonspecific binding at high cell density, apparent discrepancies between ED50′s for NADPH-oxidase and whole cell O2- generation were noted. With the use of low cell concentrations, quantitative agreement between intact cell production of O2-, NADPH-oxidase activity, and receptor binding was found. These results further support the identity of the NADPH-oxidase as the enzymatic source of respiratory burst O2- production in human neutrophils.

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Human neutrophil NADPH-oxidase activity is inhibited by lazaroids

Inflammation ◽

10.1007/bf01487401 ◽

1996 ◽

Vol 20 (2) ◽

pp. 139-150 ◽

Cited By ~ 1

Author(s):

A. J. Theron ◽

R. Anderson

Keyword(s):

Nadph Oxidase ◽

Human Neutrophil ◽

Oxidase Activity ◽

Nadph Oxidase Activity

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Ectopic Expression of an Arabidopsis Calmodulin-Like Domain Protein Kinase-Enhanced NADPH Oxidase Activity and Oxidative Burst in Tomato Protoplasts

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi.2001.14.10.1261 ◽

2001 ◽

Vol 14 (10) ◽

pp. 1261-1264 ◽

Cited By ~ 71

Author(s):

Tim Xing ◽

Xiao-Jing Wang ◽

Kamal Malik ◽

Brian L. Miki

Keyword(s):

Plasma Membrane ◽

Protein Kinase ◽

Nadph Oxidase ◽

Oxidative Burst ◽

Protein Phosphatase ◽

Oxidase Activity ◽

Ectopic Expression ◽

Free System ◽

Nadph Oxidase Activity ◽

Domain Protein

Among plant defense responses to pathogen attack, the release of active oxygen species (AOS), termed the oxidative burst, may affect the attacking pathogen and the host plant cells at the infection site, thereby limiting the spread of the pathogen. Plasma membrane-associated NADPH oxidase represents a key enzyme in mediating the oxidative burst. The mechanisms of NADPH oxidase activation, however, remains unclear. Ectopic expression of AK1-6H, an Arabidopsis calmodulin-like domain protein kinase (CDPK) in tomato protoplasts enhanced plasma membrane-associated NADPH oxidase activity. Arabidopsis protein phosphatase 2A abolished this enhancement, whereas Arabidopsis dual-specificity protein tyrosine phosphatase 1 or maize protein phosphatase 1 had no effect. tMEK2MUT, a constitutively activated, mitogen-activated protein kinase kinase from tomato, did not enhance NADPH oxidase activity when overexpressed. In a cell-free system, AK1-6H moderately stimulated the NADPH oxidase activity on plasma membrane. AK1-6H, but not tMEK2MUT, also enhanced production of AOS in intact protoplasts. Our results show that ectopic expression of a heterologous CDPK can enhance NADPH oxidase activity and stimulate an oxidative burst in tomato protoplasts.

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