Intracellular localization and preassembly of the NADPH oxidase complex in cultured endothelial cells.
The phagocyte-type NADPH oxidase expressed in endothelial cells differs from the neutrophil enzyme in that it exhibits low level activity even in the absence of agonist stimulation, and it generates intracellular reactive oxygen species. The mechanisms underlying these differences are unknown. We studied the subcellular location of (a) oxidase subunits and (b) functionally active enzyme in unstimulated endothelial cells. Confocal microscopy revealed co-localization of the major oxidase subunits, i.e. gp91(phox), p22(phox), p47(phox), and p67(phox), in a mainly perinuclear distribution. Plasma membrane biotinylation experiments confirmed the predominantly (>90%) intracellular distribution of gp91(phox) and p22(phox). After subcellular protein fractionation, approximately 50% of the gp91(phox) (91-kDa band), p22(phox), p67(phox), and p40(phox) pools and approximately 30% of the p47(phox) were present in the 1475 x g ("nucleus-rich") fraction. Likewise, approximately 50% of total NADPH-dependent O(2)() production (assessed by lucigenin (5 microm) chemiluminescence) was found in the 1475 x g fraction. Co-immunoprecipitation studies and measurement of NADPH-dependent reactive oxygen species production (cytochrome c reduction assay) demonstrated that p22(phox), gp91(phox), p47(phox), p67(phox), and p40(phox) existed as a functional complex in the cytoskeletal fraction. These results indicate that, in contrast to the neutrophil enzyme, a substantial proportion of the NADPH oxidase in unstimulated endothelial cells exists as a preassembled intracellular complex associated with the cytoskeleton.
Li
Jian-Mei
JM
Department of Cardiology, Guy's King's & St. Thomas's School of Medicine, King's College London, London SE5 9PJ, United Kingdom.
Shah
Ajay M
AM
D000818:Animals; D019921:Biotinylation; D002478:Cells, Cultured; D003599:Cytoskeleton / Q000378:metabolism; D003902:Detergents / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000166:cytology*; D018922:HL-60 Cells; D006801:Humans; D015151:Immunoblotting; D008562:Membrane Glycoproteins / Q000096:biosynthesis; D026901:Membrane Transport Proteins; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D009249:NADP / Q000378:metabolism; D009252:NADPH Dehydrogenase / Q000096:biosynthesis; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases / Q000096:biosynthesis* / Q000737:chemistry* / Q000378:metabolism; D009504:Neutrophils / Q000378:metabolism; D017830:Octoxynol / Q000494:pharmacology; D010100:Oxygen / Q000378:metabolism; D010750:Phosphoproteins / Q000096:biosynthesis; D011233:Precipitin Tests; D015398:Signal Transduction; D013347:Subcellular Fractions; D013552:Swine; D013997:Time Factors; D014471:Umbilical Veins / Q000166:cytology
D003902:Detergents; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D010750:Phosphoproteins; C079789:neutrophil cytosol factor 67K; D009249:NADP; D017830:Octoxynol; C056558:CYBB protein, human; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases; C086986:CYBA protein, human; C060505:neutrophil cytosolic factor 1; D009252:NADPH Dehydrogenase; D010100:Oxygen