scholarly journals The cytochrome b-558 molecules involved in the fibroblast and polymorphonuclear leucocyte superoxide-generating NADPH oxidase systems are structurally and genetically distinct

1993 ◽  
Vol 289 (2) ◽  
pp. 481-486 ◽  
Author(s):  
B Meier ◽  
A J Jesaitis ◽  
A Emmendörffer ◽  
J Roesler ◽  
M T Quinn
Keyword(s):  
Nadph Oxidase ◽  
Cytochrome B ◽  
Human Fibroblasts ◽  
Immunoblot Analysis ◽  
Genetic Mutation ◽  
Polymorphonuclear Leucocyte ◽  
Polymorphonuclear Leucocytes ◽  
Control Fibroblast ◽  
Release Rates ◽  
Healthy Persons

We have demonstrated that human fibroblasts can release O2-. radicals by an NADPH oxidase system that appears to be functionally similar to the phagocytic system. Further analysis of these systems, however, with respect to the low-potential b-type cytochromes involved suggests that these two O2-.-generating systems are not structurally identical. Immunoblot analysis of fibroblast membranes with six different antibodies directed against both subunits of human neutrophil cytochrome b-558 indicated that the b-type cytochrome molecules involved in these systems were not identical. None of these anti-(neutrophil cytochrome b) antibodies recognized a similar cytochrome in fibroblast membranes, suggesting that the two cytochrome species are immunologically distinct. In addition, fibroblasts obtained from a patient suffering from X-linked chronic granulomatous disease (CGD) had a normal cytochrome b-558 content compared with control fibroblast membranes, whereas the cytochrome b-558 concentration in polymorphonuclear leucocytes (PMNs) from this patient was decreased to 10% of that found in PMNs from healthy controls. Likewise, the stimulated O2-. release in PMNs from this patient was less than 10% of that in control PMNs, whereas the fibroblasts showed stimulated O2-.-release rates that were indistinguishable from those of fibroblasts obtained from healthy persons. Since the genetic mutation responsible for this type of CGD results in the absence of cytochrome b-558 in PMNs, fibroblasts should be affected in the same way if both cytochrome species were identical. These results suggest therefore that the low-potential b-type cytochromes in PMNs and fibroblasts are structurally and genetically distinct.

scholarly journals Superoxide-forming NADPH oxidase preparation of pig polymorphonuclear leucocyte

1982 ◽  
Vol 205 (3) ◽  
pp. 593-601 ◽  
Author(s):  
H Wakeyama ◽  
K Takeshige ◽  
R Takayanagi ◽  
S Minakami
Keyword(s):  
Nadph Oxidase ◽  
Enzyme System ◽  
Polymorphonuclear Leucocyte ◽  
Tween 20 ◽  
Polymorphonuclear Leucocytes ◽  
Resting Cells ◽  
Superoxide Formation ◽  
Km Value ◽  
Nadph Oxidation ◽  
Relationship Of

A phagocytic vesicle fraction with high NADPH-dependent superoxide-forming activity was obtained in large quantity from pig blood polymorphonuclear leucocytes, phagocytosing oil droplets in the presence of cyanide. The activity of the homogenate of the phagocytosing cells was 40 times that of the resting cells, and 70% of the activity in the homogenate was recovered in the phagocytic vesicle fraction. Essentially all of the superoxide-forming activity was extracted by repeated extraction with a mixture containing deoxycholate and Tween 20. The extract had a superoxide-forming activity of 1 mumol/min per mg of protein with NADPH, and one-fifth of this with NADH, Km values being similar to those of the vesicle fraction (40 microM for NADPH and 400 microM for NADH). A stoichiometric relationship of 1:2 for NADPH oxidation and superoxide formation was obtained, in agreement with the reaction NADPH +2O2 leads to NADP+ + 2O2 -. + H+. The activity of the extract was enhanced 2-fold by the addition of FAD, suggesting that the flavin is a component of the enzyme system. The Km value for FAD was 0.077 microM. The activities in both vesicle fraction and extract were labile even on refrigeration, but could be kept for several months at −70 degrees C.

Protein kinase C-α and -δ are required for NADPH oxidase activation in WKYMVm-stimulated IMR90 human fibroblasts

2007 ◽  
Vol 459 (2) ◽  
pp. 288-294 ◽  
Author(s):  
Annalisa Iaccio ◽  
Claudio Collinet ◽  
Nicola Montesano Gesualdi ◽  
Rosario Ammendola
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Nadph Oxidase ◽  
Human Fibroblasts ◽  
Kinase C ◽  
Nadph Oxidase Activation

scholarly journals Studies on the electron-transfer mechanism of the human neutrophil NADPH oxidase

1989 ◽  
Vol 262 (2) ◽  
pp. 575-579 ◽  
Author(s):  
J A Ellis ◽  
A R Cross ◽  
O T G Jones
Keyword(s):  
Electron Transfer ◽  
Steady State ◽  
Nadph Oxidase ◽  
Cytochrome B ◽  
Human Neutrophil ◽  
Sodium Deoxycholate ◽  
Human Neutrophils ◽  
Electron Transfer Mechanism ◽  
State Reduction ◽  
O2 Production

A superoxide-generating NADPH oxidase was solubilized from phorbol 12-myristate 13-acetate-activated human neutrophils with a mixture of sodium deoxycholate (0.125%, w/v) and Lubrol-PX (0.125%, v/v). The solubilized preparation contained FAD (577 pmol/mg of protein) and cytochrome b-245 (479 pmol/mg of protein) and produced 11.61 mol of O2-./s per mol of cytochrome b (340 nmol of O2-./min per mg of protein). On addition of NADPH, the cytochrome b-245 was reduced by 7.9% and the FAD by 38% in the aerobic steady state; NADH addition caused little steady-state reduction of cytochrome b and FAD. In this preparation, and several others, the measured rate of O2-. production correlated with the turnover of cytochrome b calculated from the extent of cytochrome b-245 reduction under aerobic conditions. Addition of diphenyleneiodonium abolished the reduction of both the FAD and cytochrome b-245 components and inhibited O2-. production. The haem ligand imidazole inhibited O2-. generation and cytochrome b reduction while permitting FAD reduction. These results support the suggestion that the human neutrophil NADPH oxidase has the electron-transport sequence: NADPH-FAD-cytochrome b-245-O2.

scholarly journals Subcellular localization of NAD(P)H oxidase(s) in human neutrophilic polymorphonuclear leucocytes

1978 ◽  
Vol 176 (1) ◽  
pp. 175-178 ◽  
Author(s):  
D B Iverson ◽  
P Wang-Iverson ◽  
J K Spitznagel ◽  
L R DeChatelet
Keyword(s):  
Cell Membrane ◽  
Nadph Oxidase ◽  
Subcellular Localization ◽  
Density Gradient ◽  
Membrane Fraction ◽  
Sucrose Density Gradient ◽  
Human Neutrophils ◽  
Polymorphonuclear Leucocytes

NADH and NADPH oxidase activities in a homogenate of human neutrophils co-sediment in a linear sucrose density gradient under either velocity or isopycnic conditions of centrifugation. The position of these activities in the gradient does not correspond to any known subcellular granule or to the cell-membrane fraction. These data suggest that the oxidase activities may reside in a unique granule that has previously not been recognized.

Abstract 352: Anti-aging Gene Klotho Deficiency Causes Hypertension and Vascular Dysfunction via Upregulation of Mammalian Target of Rapamycin (mTOR)

Hypertension ◽  
2012 ◽  
Vol 60 (suppl_1) ◽  
Author(s):  
Ying Song ◽  
Zhongjie Sun
Keyword(s):  
Blood Pressure ◽  
Nadph Oxidase ◽  
Oxidase Activity ◽  
Aging Process ◽  
Vascular Dysfunction ◽  
Mammalian Target Of Rapamycin ◽  
Genetic Mutation ◽  
Oxidase Inhibitor ◽  
Nadph Oxidase Activity ◽  
Induced Hypertension

Klotho is a recently discovered anti-aging gene. Genetic mutation of klotho expedites the aging process and shortens the lifespan while overexpression of klotho slows down the aging process and extends the lifespan by 20%. Interestingly, blood pressure (BP) was elevated significantly and vasodilatory responses to acetylcholine and sodium nitroprusside were impaired in klotho heterozygeous (+/-) mice, suggesting that klotho deficiency causes hypertension and vascular dysfunction. It is noted that klotho deficiency is associated with upregulation of mTOR expression and NADPH oxidase activity and downregulation of Mn-SOD expression in aortas and kidneys. Inhibition of mTOR by rapamycin abolished the upregulation of NADPH oxidase activity and O 2 - production and the downregulation of Mn-SOD expression and decreased BP to the control levels. Inhibition of mTOR also abolished vascular endothelial dysfunction and macrophage infiltration in kidneys in klotho (+/-) mice. The upregulation of NADPH oxidase activity and downregulation of Mn-SOD may be involved in klotho deficiency-induced hypertension which can be decreased significantly by apocynin (NADPH oxidase inhibitor) or Tempol (O 2 - scavenger). These results demonstrate, for the first time, that klotho is essential in the maintenance of normal blood pressure. Klotho deficiency-induced hypertension and vascular dysfunction are mediated by upregulation of mTOR. This study also reveals a previously unidentified role of mTOR in the regulation of NADPH oxidase and MnSOD. (Supported by HL105302 and HL102074).

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