Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:1.6.3.1 (NADPH oxidase)
 11,281 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In order to resolve discrepancies in the literature concerning the subcellular localization of NADPH oxidase, we disrupted human neutrophils by nitrogen cavitation and fractionated the subcellular organelles on a discontinuous sucrose density gradient. The lightest fraction was 20- to 40-fold enriched for plasma membranes as determined by the marker enzymes alkaline phosphatase and phosphodiesterase I as well as by the ratio of lipid phosphorus to protein. There was a significant decrease in the specific activities of the granule markers myeloperoxidase, lysozyme, and beta-glucuronidase. An intermediate fraction was enriched in membrane markers but not to the extent the lightest fraction was enriched. This fraction contained more granular contamination, as shown by the marker enzymes. In contrast, the densest bands of the gradient were enriched for granule markers with little contamination by plasma membrane. Superoxide generation and NADP formation were primarily associated with the two membrane-enriched fractions from polymorphonuclear leukocytes stimulated with phorbol myristate acetate. The NADP formation associated with a dense granule fraction observed previously in our laboratory was probably due to a cyanide-stimulated oxidation of NADPH by myeloperoxidase.
Inflammation 1984 Sep
PMID:Co-localization of superoxide generation and NADP formation in plasma membrane fractions from human neutrophils. 609 76

A membrane-bound NADPH-cytochrome c reductase, which is capable of forming the superoxide anion (O2-) in the presence of menadione, was highly purified from membrane fractions of disrupted guinea pig polymorphonuclear leukocytes by solubilization with 0.2% Triton X-100 and chromatographies on Sephacryl S-300 and 2',5'-ADP-agarose. The overall purification from the membrane fraction was over 110-fold, with a yield of about 6%. The purified preparation did not contain two other pyridine nucleotide-oxidizing enzymes: NADH- and NAD(P)H-oxidizing enzymes (J. Biochem. 94, 931-936, 1983). Besides cytochrome c, the purified enzyme was able to reduce menadione, Nitroblue tetrazolium (NBT) and 2,6-dichlorophenolindophenol. The reduction of menadione alone resulted in the formation of O2-. The purified enzyme preparation contained FAD. When assayed by measuring O2--generation in the presence of menadione, the enzyme showed an optimum pH at 7.0-7.4, and Km values for NADPH, NADH, and menadione were 25, 230, and 5.3 microM, respectively. The enzyme activity was not inhibited by NaN3 or dicumarol, but was by N-ethylmaleimide, EDTA, and quercetin; these inhibition profiles agree with those observed for the NADPH oxidase in the membrane fraction of phorbol-myristate acetate-stimulated leukocytes. Furthermore, when compared by means of the NBT-staining method combined with disc gel electrophoresis, the purified enzyme was electrophoretically indistinguishable from the NADPH-NBT reductase in the plasma membrane as well as phagosomes of the leukocytes. These results suggest that the purified NADPH-cytochrome c reductase is the putative flavoprotein of the NADPH oxidase system responsible for the respiratory burst.
J Biochem 1984 Sep
PMID:Purification and characterization of a membrane-bound NADPH-cytochrome c reductase capable of catalyzing menadione-dependent O2- formation in guinea pig polymorphonuclear leukocytes. 609 21

Kupffer cells were isolated from pronase-perfused rat livers and were maintained as a monolayer culture in a state of high purity and viability. Immediately after contact with zymosan particles, O2 uptake of the Kupffer cells increased fivefold; about 50% of the net oxygen consumed was accounted for as superoxide released into the medium. Concomitantly, a transient burst of luminol-dependent chemiluminescence, an increased activity of NAD(P)H oxidase and a stimulation of the flow of glucose through the hexose monophosphate shunt were observed. Chemiluminescence and O2- production were almost completely inhibited by superoxide dismutase and iodoacetate. Zymosan-induced chemiluminescence was not inhibited in the presence of the non-penetrating thiol reagents, 5,5'-dithio-bis-2-nitrobenzoate and iodoacetyl-sepharose. Iodoacetate acted on the cytosolic glucose-6-phosphate dehydrogenase rather than on NAD(P)H oxidase of the cell membrane.
Eur J Biochem 1981 Sep
PMID:Superoxide release by zymosan-stimulated rat Kupffer cells in vitro. 628 Oct 2

Mouse peritoneal macrophages respond to environmental stimuli in different ways depending on their state of differentiation. Macrophages from mice with bacillus Calmette--Guerin (BCG) infection produced large amounts of H2O2 in response to phorbol diesters (PDEs), while those from noninfected mice produced little or no H2O2. The effects of PDEs on cells are mediated by specific cellular receptors for these ligands. The purpose of this study was to determine if the varying responses of macrophages from different groups of mice were caused by differences in their receptors for the PDE ligands. By all parameters studied, the binding of [20-3H]phorbol 12,13-dibutyrate ( [3H]PDBu) was similar in all macrophages irrespective of their ability to produce H2O2 in response to PDEs. Binding of [3H]PDBu was rapid at 23 degrees C reaching a maximum at 10-20 min with a subsequent decline to 50-60% of maximum by 30-60 min. Binding was slower at 0 degrees C reaching a maximum at 90-120 min. The binding was reversible, with dissociation kinetics paralleling association kinetics. The binding was saturable; the Kd's (45 to 91 nM) and number of binding sites (about 7-14 X 10(5)/cell or 11-12 pmol/mg protein) were essentially the same for the different classes of macrophages. The binding was specific, and analogs of PDBu inhibited [3H]PDBu binding to macrophages with potencies comparable to their potencies in causing in vivo tumor promotion and elicitation of other cellular responses in vitro. The ligands [3H]PDBu and [3H]PMA were degraded to comparable degrees by macrophages from normal or BCG-infected mice. Macrophages from C3H/HeJ and C3H/HeN mice, although known to differ in their abilities to respond to stimuli such as lymphokines and LPS, did not differ in their ability to produce H2O2 in response to PDEs or in their receptors for PDEs. Results of this study suggest that in vivo "activation" of macrophages in mice infected with BCG is not associated with a change in the cells' receptors for PDEs, but may be associated with "postreceptor" changes such as linkage of the PDE receptor with NAD(P)H oxidase, a change in NAD(P)H oxidase, or induction of synthesis of NAD(P)H oxidase.
Cell Immunol 1983 Sep
PMID:Phorbol diester-induced H2O2 production by peritoneal macrophages. Different H2O2 production by macrophages from normal and BCG-infected mice despite comparable phorbol diester receptors. 630 16

Guinea pig polymorphonuclear leukocytes (PMN) were briefly activated with soluble stimulators such as sodium myristate (SM) or phorbol myristate acetate (PMA) and then disrupted by the nitrogen cavitation method to study the subcellular distribution of NADPH oxidase, which is responsible for O2 - generation. Fc-receptor and 5'-nucleotidase activities were measured as plasma membrane markers. 1) The homogenate was first fractionated by differential centrifugation. The O2- -generating activity of PMN activated either by SM or PMA was recovered in a 2 X 10(4) g pellet which contained a large amount of granules and about 50% of the plasma membrane markers, but not in a 1 X 10(5) g pellet which consisted of plasma membranes and few granules. 2) Further separation of the 2 X 10(4) g pellet from PMA-activated PMN was attempted by an iso-osmotic Percoll density gradient centrifugation. The O2- -generating activity was recovered in light fractions in which plasma membrane markers were found, but neither in specific nor in azurophil granules. The 1 X 10(5) g pellet showed a similar distribution of the plasma membrane markers to that of the 2 X 10(4) g pellet, except that the peak of the O2- -generating activity was much smaller on an identical density gradient. The results showed that NADPH oxidase is located in the plasma membranes precipitated by centrifugation at 2 X 10(4) X g but not in the ones precipitated at 1 X 10(5) X g. The results suggest that the plasma membrane of activated PMN has a mosaic distribution of NADPH oxidase.
J Biochem 1983 Sep
PMID:Activation of guinea pig polymorphonuclear leukocytes with soluble stimulators leads to nonrandom distribution of NADPH oxidase in the plasma membrane. 631 91

The phosphorylation of proteins in intact pig polymorphonuclear leukocytes loaded with H3(32)PO4 was investigated by two-dimensional gel electrophoresis and subsequent autoradiography. The incorporation of 32P into at least 17 proteins began to increase and into one to decrease, relative to resting cells, upon exposure of the cells to phorbol 12-myristate 13-acetate. These changes in the autoradiographic patterns were accompanied by changes in the protein patterns obtained by staining with Coomassie brilliant blue, including the appearance, the acidic shift and the increase or decrease of the intensity of the spots. Among these proteins, Mr = 64 000, 31 000, 22 000, 21 000, 18 000 and 13 000 proteins were correlated well with the superoxide anion production of the cells in respect to the time-courses and the dose-responses. By taking the effects of EGTA into consideration, the phosphorylation of Mr 64 000 and 21 000 proteins, of which the latter was identified as the light chain of myosin, seemed to be involved in the signal-transmission mechanism of the induction of the NADPH oxidase responsible for the 'respiratory burst'. These two proteins were also phosphorylated in the cells stimulated by NaF or oil droplets opsonized with IgG.
Biochim Biophys Acta 1984 Sep 14
PMID:Protein phosphorylation in intact pig leukocytes. 647 72

1. The luminol-dependent chemiluminescence of rat thymocytes responding to concanavalin A can be resolved into glucose-dependent and glucose-independent portions. 2. The glucose-dependent portion, supported by D-glucose and D-mannose oxidation, is inhibited by catalase (200 microgram/ml), amobarbital (1 mM) and hexose analogues that block D-glucose uptake. Thus concanavalin A may activate, transiently, an NAD(P)H oxidase that utilizes reducing equivalents derived from the oxidation of exogenous glucose to give dismutation products of O2- (including H2O2) as its major products. 3. The glucose-independent portion is inhibited by eicosa-5,8,11,14-tetraynoic acid but not by indomethacin. It may therefore be associated with the conversion of hydroperoxy intermediates of arachidonic acid metabolism to hydroxy products by the lipoxygenase pathway. 4. Preincubation of thymocytes for 18 h in serum-free medium enhances the subsequent chemiluminescent response to concanavalin A severalfold and evokes the response at a lower threshold concentration. The incorporation of [3H]thymidine by preincubated cells is similarly enhanced at low doses of concanavalin A, whereas the response to optimal doses is unaltered. 5. Catalase does not inhibit the enhanced incorporation of [3H]thymidine obtained in response to concanavalin A, but instead amplifies the response to low doses in the same manner as preincubation.
Biochem J 1981 Sep 15
PMID:Concanavalin A-induced chemiluminescence in rat thymus lymphocytes. Its origin and role in mitogenesis. 697 84

NAD(P)H oxidase activity was determined in particulate fractions from human neutrophils by measuring the production of hydrogen peroxide. Activity was measured over a wide range of substrate concentrations from 0.0 to 4.0 mM. The activity with NADPH was consistently greater than with NADH. Activity towards both substrates was higher in a particulate fraction derived from cells which had phagocytized opsonized zymosan than in a corresponding fraction from resting cells. This increased activity was apparently due to a decreased Km of the enzyme, although no evidence of allosteric kinetics was obtained. The activity was markedly reduced in the presence of superoxide dismutase, indicating the involvement of a superoxide-mediated chain reaction. Particular fractions derived from cells of a patient with chronic granulomatous disease exhibited decreased activity towards both substrates and an apparent defect in the activation of the enzyme by phagocytosis.
Inflammation 1982 Sep
PMID:Pyridine nucleotide-dependent generation of hydrogen peroxide by a particulate fraction from human neutrophils: effect of substrate concentration. 712 96

The aim of this study was to determine the cellular source of oxygen free radicals generated by isolated hepatocytes during post-anoxic reoxygenation. Superoxide anions (O2.-) were detected by lucigenin chemiluminescence. Cell damage was assessed by LDH release. During anoxia, the chemiluminescence decreased to background levels while LDH release increased 8-fold. During reoxygenation, O2.- formation increased 15-fold within 15 min then declined towards control levels. LDH release increased from 161 to 285 mU/min in the first 30 min of reoxygenation, then declined toward the control rate. Allopurinol, an inhibitor of the xanthine-xanthine oxidase system, did not inhibit O2.- formation nor LDH release. Antimycin, a mitochondrial complex III inhibitor that does not block O2.- formation, increased both O2.- generation and LDH release 82 and 133% respectively. Diphenyleneiodonium (DPI), a mitochondrial and microsomal NADPH oxidase inhibitor, reduced O2.- and LDH release 60-70%. SOD, which catalyzes the dismutation of O2.- to H2O2, was without effect on O2.- and LDH release, but TEMPO, a stable nitroxide which mimics SOD and easily penetrates the cell membrane, decreased O2.-86% without affecting LDH. These results suggest that mitochondria or microsomes are the principal sites of O2.- production during reoxygenation of isolated hepatocytes, whereas the cytosolic xanthine/xanthine oxidase system is apparently not involved.
Biochim Biophys Acta 1995 Sep 21
PMID:Source of oxygen free radicals produced by rat hepatocytes during postanoxic reoxygenation. 754 22

Administration of single doses of 0.1 mg L-triiodothyronine (T3)/kg for 3 consecutive days to fed rats produced a drastic increase in the respiratory burst activity of isolated polymorphonuclear leukocytes (PMN), stimulated with serum-opsonized zymosan. This effect was evidenced by the 3.8-fold increment in the integrated chemiluminescence, and seems to be primarily related to the enhanced activity of NADPH oxidase elicited by T3 treatment, with the observed higher myeloperoxidase activity playing a contributory role. In these conditions, hyperthyroidism determines a net enhancement in the oxidant capacity of PMN, as the increased rate of O2.- generation found occurs in the absence of changes in the activity of superoxide dismutase.
Free Radic Biol Med 1995 Sep
PMID:On the mechanism of thyroid hormone-induced respiratory burst activity in rat polymorphonuclear leukocytes. 755 50


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