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PubMed1993

Purification and immunohistochemical tissue localization of human xanthine oxidase.

Xanthine oxidase was purified 1600-fold from human liver cytosol. The purified enzyme was shown as a single band of 300 kDa on polyacrylamide gel electrophoresis and 150 kDa on SDS-PAGE. Using this purified enzyme, polyclonal antibody against xanthine oxidase was raised in a rabbit. On Ouchterlony's double immunodiffusion method, the raised antibody and the human liver cytosol made a precipitation line stained by activity stain and protein stain, respectively. With the raised anti-xanthine oxidase sera, the immunohistochemical localization of xanthine oxidase in human tissues was examined. Immunostaining of frozen hepatic tissue section showed that the cytoplasm of hepatocytes and endothelial lining cells were stained. In a number of other tissues, the xanthine oxidase antigen was detected only in the endothelial lining cells from heart, kidney, brain, aorta, lung and mesentery, except for the duodenal mucosa cells. A possible role for xanthine oxidase in the endothelial cells from various human tissues in the pathogenesis of reperfusion injury was suggested.

Antibody Specificity↗
PubMed1994

Inhibition of thyroid NADPH-oxidase by 2-iodohexadecanal in a cell-free system.

The major nonpolar iodolipid formed in horse thyroid cells has recently been identified as 2-iodohexadecanal (2-IHDA). We have investigated in vitro the effect of 2-IHDA on the NADPH-oxidase, NADPH-cytochrome c reductase, and thyroid peroxidase (TPO) activities of a porcine thyroid plasma membrane preparation. 2-IHDA inhibited NADPH-oxidase activity, with half-inhibition at 3-5 microM, but it had no effect on NADPH-cytochrome c reductase. It inhibited the TPO-catalyzed iodination of protein, but not iodide oxidation. Hexadecanal also inhibited NADPH-oxidase. Inhibition by the non-iodinated lipid aldehydes depended on the length of their aliphatic chain: dodecanal and tridecanal gave maximal inhibition. Free iodide, 2-iodohexadecanol and palmitic acid all had no inhibitory effect. Washing treated membranes showed that the inhibition of NADPH-oxidase by hexadecanal was fully reversible, whereas that of 2-IHDA and other iodinated or brominated alkanals was irreversible. Thus the interaction between some residues of the thyroid NADPH-oxidase and the lipid aldehyde groups was favored or stabilized by the iodine atom. Modification of primary amine and thiol groups of NADPH-oxidase inhibited its activity. These groups could also be the target of lipid aldehydes. We suggest that 2-IHDA, because it inhibits TPO and more profoundly the H2O2-generating system in thyroid plasma membrane, modulates iodide metabolism in the thyrocyte and may mediate the Wolff-Chaikoff effect.

Aldehydes↗
PubMed1980

L-gulonolactone oxidase activity and vitamin C status in riboflavin-deficient rats.

The effect of riboflavin deficiency on the activity of L-gulonolactone oxidase [L-gulono-gamma-lactone: oxygen 2-oxidoreductase, EC 1.1.3.8] and on vitamin C status was studied. A marked decrease in the specific activity of L-gulonolactone oxidase was observed in the liver microsomes isolated from riboflavin-deficient rats: the specific activity was approx. one-third of that in the microsomes isolated from control rats. The L-ascorbic acid content in the liver of the riboflavin-deficient rats was approx. one-half of that in the liver of the control rats. It seems that the rate of production of L-ascorbic acid in the riboflavin-deficient rats is limited by the decreased level of L-gulonolactone oxidase activity. Immunotitration using rabbit antiserum directed to L-gulonolactone oxidase revealed that a substantial amount of an inactive form of this enzyme is present in the liver microsomes of the riboflavin-deficient rats. L-Gulonolactone oxidase activity in the microsomes of these rats increased by approx. 35% upon addition of FAD, but it was slightly decreased by the addition of FMN or riboflavin. These results indicate that the liver microsomes of the riboflavin-deficient rats contain a protein which exhibits L-gulonolactone oxidase activity upon addition of Fad.

Animals↗
PubMed1984

Ontogenetic development of L-gulonolactone oxidase activity in several vertebrates.

Activity of L-gulonolactone oxidase (EC 1.1.3.8) in livers of fetal Rattus norvegicus and Mus musculus was detectable on the 18th day of gestation, increased rapidly to maxima at 15 and 5 days postpartum for the two species, respectively, and thereafter declined to adult levels. L-Gulonolactone oxidase was not detectable in liver or kidney of fetal guinea pigs at any stage of development. Near-term fetal snowshoe hares had higher activities of liver L-gulonolactone oxidase than observed in a large sample of adults. L-Gulonolactone oxidase was detectable in chicken (Gallus gallus) embryos by the sixth day of incubation, increased rapidly in the kidney with no discontinuity at hatching, reached a maximum at about the 35th day from the beginning of incubation, and then declined to adult levels. Barn swallow (Hirundo rustica) embryos appeared to synthesize little if any L-ascorbic acid; nestlings had considerably higher levels of L-gulonolactone oxidase than adults. Tadpoles of three species of frogs had appreciable levels of L-gulonolactone oxidase activity.

Aging↗
PubMed1995

Anti-inflammatory action of gentamycin through inhibitory effect on neutrophil NADPH oxidase activity.

The effects of gentamycin on the NADPH oxidase (EC 1.6.99.6) from human neutrophils in both whole-cell and fully soluble (cell-free) systems were investigated. Gentamycin was found to inhibit, concentration-dependently, the superoxide generation of neutrophils exposed to phorbol myristate acetate in a whole-cell system and the activation of superoxide-generating NADPH oxidase by sodium dodecyl sulfate in a cell-free system. The concentrations of the drug required for 50% inhibition of the oxidase (IC50) were 150 microM in the whole-cell system and 10 microM in the cell-free system. In addition, in the cell-free system, the drug did not change the Km value for NADPH of the oxidase. However, gentamycin did not the superoxide generation of NADPH oxidase after its activation in the cell-free system, suggesting that the drug do not have superoxide-scavenger action. These results suggest that gentamycin, an aminoglycoside antibiotic, may exhibit an anti-inflammatory action due to inhibition of neutrophil NADPH oxidase activation.

Anti-Inflammatory Agents, Non-Steroidal↗
PubMed1989

Demonstration of monoamine oxidase-A and -B in the human brainstem by a histochemical technique.

The distribution of both monoamine oxidase subtypes, monoamine oxidase-A and -B, is demonstrated in brainstems from 16 humans by use of a histochemical technique. The results presented here, focus primarily upon the aminergic areas of the substantia nigra, the locus coeruleus and the raphe nuclei. While dopaminergic neurons of the substantia nigra revealed no staining for monoamine oxidase, noradrenergic neurons of the locus coeruleus stained positively with the monoamine oxidase-A substrate serotonin, and serotonergic neurons of the raphe nuclei were stained by the monoamine oxidase-B substrate beta-phenylethylamine. In addition, data are presented showing that glial cells stain predominantly for monoamine oxidase-B.

Adolescent↗
PubMed1991

Molecular cloning of human lysyl oxidase and assignment of the gene to chromosome 5q23.3-31.2.

Lysyl oxidase (EC 1.4.3.13) initiates the crosslinking of collagens and elastin by catalyzing oxidative deamination of the epsilon-amino group in certain lysine and hydroxylysine residues. We report here on the isolation and characterization of cDNA clones for the enzyme from human placenta and rat aorta lambda gt11 cDNA libraries. A cDNA clone for human lysyl oxidase covers all the coding sequences, 230 nucleotides of the 5' and 299 nucleotides, of the 3' untranslated sequences, including a poly(A) tail of 23 nucleotides. This cDNA encodes a polypeptide of 417 amino acid residues, including a signal peptide of 21 amino acids. Sequencing of two rat lysyl oxidase cDNA clones indicated six differences between the present and the previously published sequence for the rat enzyme [Trackman et al. (1990) Biochemistry 29: 4863-4870], resulting in frameshifts in the translated sequence. The human lysyl oxidase sequence was found to be 78% identical to the revised rat sequence at the nucleotide level and 84% identical at the amino acid level, with the degree of identity unevenly distributed between various regions of the coded polypeptide. Northern blot analysis of human skin fibroblasts RNA indicated that the human lysyl oxidase cDNA hybridizes to at least four mRNA species; their sizes are about 5.5, 4.3, 2.4, and 2.0 kb. Analysis of a panel of 25 human x hamster cell hybrids by Southern blotting mapped the human lysyl oxidase gene to chromosome 5, and in situ hybridization mapped it to 5q23.3-31.2.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

A sensitive fluorometric assay for measuring xanthine dehydrogenase and oxidase in tissues.

The conversion of xanthine dehydrogenase to a free radical producing oxidase is an important component of oxygen-mediated tissue injury. Current assays for these enzymes are of limited sensitivity, making it difficult to analyze activities in organ biopsies or cultured cells. The xanthine oxidase-catalyzed conversion of pterin (2-amino-4-hydroxypteridine) to isoxanthopterin provides the basis for a fluorometric assay which is 100-500 times more sensitive than the traditional spectrophotometric assay of urate formation from xanthine. Enzyme activity as low as 0.1 pmol min-1 ml-1 can be measured with the fluorometric pterin assay. Xanthine oxidase is assayed in the presence of pterin only, while combined xanthine dehydrogenase plus oxidase activity is determined with methylene blue which replaces NAD+ as an electron acceptor. The relative proportions and specific activities of xanthine oxidase and dehydrogenase determined by the fluorometric pterin assay are comparable with the spectrophotometric measurement of activities present in rat liver, intestine, kidney, and plasma. The assay has been successfully applied to brain, human kidney, and cultured mammalian cells, where xanthine dehydrogenase and oxidase activities are too low to detect spectrophotometrically.

Allopurinol↗
PubMed1990

Ethanol-induced iron mobilization: role of acetaldehyde-aldehyde oxidase generated superoxide.

Superoxide radicals, a species known to mobilize ferritin iron, and their interaction with catalytic iron have been implicated in the pathogenesis of alcohol-induced liver injury. The mechanism(s) by which ethanol metabolism generates free radicals and mobilizes catalytic iron, however, is not fully defined. In this investigation the role of hepatic aldehyde oxidase in the mobilization of catalytic iron from ferritin was studied in vitro. Iron mobilization due to the metabolism of ethanol to acetaldehyde by alcohol dehydrogenase was increased 100% by the addition of aldehyde oxidase. Iron release was favored by low pH and low oxygen concentration. Mobilization of iron due to acetaldehyde metabolism by aldehyde oxidase was completely inhibited by superoxide dismutase but not by catalase suggesting that superoxide radicals mediate mobilization. Acetaldehyde-aldehyde oxidase mediated reduction of ferritin iron was facilitated by incubation with menadione, an electron acceptor for aldehyde oxidase. Mobilization of ferritin iron due to the metabolism of acetaldehyde by aldehyde oxidase may be a fundamental mechanism of alcohol-induced liver injury.

Acetaldehyde↗
PubMed1995

Effects of fatty acids and fatty acyl CoA esters on Cu(2+)-induced conversion of xanthine dehydrogenase to oxidase in rabbit liver.

Effects of various fatty acids and fatty acyl CoA esters on Cu(2+)-induced conversion of xanthine dehydrogenase to oxidase in rabbit liver were examined. Cu2+ (2-10 microM) brought about the conversion of xanthine dehydrogenase to oxidase in a dose-dependent manner. Oleic, arachidonic, eicosapentaenoic, and docosahexaenoic acids (50-200 microM) prevented the conversion of xanthine dehydrogenase to oxidase catalyzed by 6 microM-Cu2+. The effect of these four fatty acids was concentration-dependent, whereas palmitic, stearic, and linoleic acids had no effect on the conversion of xanthine dehydrogenase to oxidase at the same concentration range. On the other hand, palmitoyl, linoleoyl, and arachidonoyl CoAs elicited the inhibition of 6 microM-Cu(2+)-induced conversion of xanthine dehydrogenase to oxidase at concentrations of 50, 100, and 200 microM. These results suggest that oleic, arachidonic, eicosapentaenoic and docosahexaenoic acids, and fatty acyl CoAs have the potential to inhibit the conversion of xanthine dehydrogenase to oxidase in rabbit liver.

Acyl Coenzyme A↗
PubMed1995

Altered kinetics of cytochrome c oxidase in a patient with severe mitochondrial encephalomyopathy.

Deficiency of cytochrome c oxidase activity was established in a girl born to consanguineous parents. She showed symptoms of dysmaturity, generalized hypotonia, myoclonic seizures and progressive respiratory failure, leading to death on the seventh day of life. Structural abnormalities of the central nervous system consisted of severe cerebellar hypoplasia and optic nerve atrophy. Biochemical analysis of a muscle biopsy specimen demonstrated deficiency of cytochrome c oxidase activity. Cultured fibroblasts from this patient also showed a selective decrease in the activity of cytochrome c oxidase, excluding a muscle-specific type of deficiency. Further investigations in cultured fibroblasts revealed that synthesis, assembly and stability of both the mitochondrial and the nuclear subunits of the enzyme were entirely normal. The steady-state concentration of cytochrome c oxidase in the fibroblasts of the patient was also normal, suggesting that the kinetic properties of the enzyme were altered. Analysis of the kinetic parameters of cytochrome c oxidase demonstrated an aberrant interaction between cytochrome c oxidase and its substrate, cytochrome c, most likely because of a mutation in one of the nuclear subunits of the enzyme.

Cells, Cultured↗
PubMed2004

Phosphoinositide 3-kinase regulates the phosphorylation of NADPH oxidase component p47(phox) by controlling cPKC/PKCdelta but not Akt.

Superoxide production by NADPH oxidase is essential for the bactericidal properties of phagocytes. Phosphorylation of p47(phox), one of the cytosolic components of NADPH oxidase, is a crucial step of the oxidase activation. Some evidences suggest that phosphoinositide 3-kinase (PI3K) is involved in p47(phox) phosphorylation, but it has not been fully understood how PI3K regulates it. The aim of this study was to examine the mechanism underlying the PI3K regulation of p47(phox) phosphorylation. Pharmacological inhibition of PI3K attenuated both fMLP-stimulated p47(phox) phosphorylation and NADPH oxidase activity in HL-60 cells differentiated to a neutrophil-like phenotype. Although fMLP elicited Akt activation in a PI3K-dependent manner, an Akt inhibitor had no effect on the oxidase activity triggered by fMLP. In vitro kinase assay revealed that Akt was unable to catalyze p47(phox) phosphorylation. Interestingly, the activation of cPKC and PKCdelta after fMLP stimulation was dependent on PI3K. Furthermore, PI3K inhibitors reduced the activation of phospholipase Cgamma2 without affecting tyrosine phosphorylation on it. These results suggest that PI3K regulates the phosphorylation of NADPH oxidase component p47(phox) by controlling diacylglycerol-dependent PKCs but not Akt.

Androstadienes↗
PubMed2004

Binding of arachidonic acid to myeloid-related proteins (S100A8/A9) enhances phagocytic NADPH oxidase activation.

Activation of the O(2)(-) generating NADPH oxidase of phagocytes results from the assembly of the membrane-bound flavocytochrome b(558) with cytosolic proteins, p67(phox), p47(phox), and Rac. However, it has been recently reported that the arachidonic acid- and calcium-binding heterodimer S100A8/A9, abundant in neutrophil cytosol, influences the activation process. In a semi-recombinant system comprising neutrophil membranes, recombinant proteins, p67(phox), p47(phox), GTPgamma S-loaded Rac2, and arachidonic acid (AA), both the rate and the extent of the oxidase activation were increased by S100A8/A9, provided it was preloaded with AA. Binding of [(14)C]AA to S100A8/A9 was potentiated by recombinant cytosolic phox proteins and GTPgammaS, suggesting the formation of a complex, comprising oxidase activating proteins and S100A8/A9, with a greater affinity for AA. The rate constant of oxidase activation was not increased by AA-loaded S100A8/A9, whereas the maximal oxidase activity elicited was twice as high. AA-loaded S100A8/A9 increases oxidase activation probably by decreasing the deactivation rate.

Arachidonic Acid↗
PubMed2005

Role of alpha1-adrenoreceptors in cocaine-induced NADPH oxidase expression and cardiac dysfunction.

OBJECTIVE: We assessed whether alpha1-adrenoreceptor (alpha1-AR) stimulation contributes to activation of myocardial NADPH oxidase in a rat model of cocaine-induced cardiac dysfunction. METHODS AND RESULTS: After 7 days of cocaine injection (2 x 7.5 mg/kg/day, i.p., Coc), NADPH activity assessed by chemiluminescence increases as well as phosphorylation of p47phox, one of the cytosolic components of NADPH oxidase. The alpha1-AR antagonist prazosin (Prz), administered 1 h before each cocaine injection (2 x 1 mg/kg/day, i.p., Coc+Prz), prevents these effects. Moreover, Prz pretreatment reduces left ventricular/body weight (LV/BW) ratio and partially prevents the cocaine-induced alterations in fractional shortening and cardiac index assessed by echocardiography. In order to confirm the involvement of alpha1-AR stimulation in NADPH oxidase up-regulation in vivo, we used phenylephrine (Phe) administration with the same protocol of injections as that used with cocaine (2 x 5 microg/kg/day, i.p.). After Phe administration, as expected, NADPH oxidase activity increases as well as phosphorylation of p47phox. These effects occur in the absence of sustained hemodynamic changes. CONCLUSION: This study demonstrates the involvement of the alpha1-AR in NADPH oxidase activation and in cocaine-induced LV dysfunction. We suggest that alpha1-AR stimulation, at least in part via NADPH oxidase induction, plays a critical role in the events leading to the cardiomyopathy observed after cocaine abuse.

Adrenergic alpha-Agonists↗
PubMed2005

NADPH oxidase and apoptosis in cerulein-stimulated pancreatic acinar AR42J cells.

Apoptosis linked to oxidative stress has been implicated in pancreatitis. We investigated whether NADPH oxidase mediates apoptosis in cerulein-stimulated pancreatic acinar AR42J cells. We report here that cerulein treatment resulted in the activation of NADPH oxidase, as determined by ROS production, translocation of cytosolic subunits p 47(phox) and p 67(phox) to the membrane, and interaction between NADPH oxidase subunits. Cerulein induced Ca(2+) oscillation, the expression of apoptotic genes p53 and bax, and apoptotic indices (DNA fragmentation, TUNEL staining, caspase 3 activity, decrease in cell viability) in AR42J cells. Treatment with a Ca(2+) chelator, BAPTA-AM, or transfection with antisense oligonucleotides for NADPH oxidase subunits p22(phox) and p 47(phox) inhibited cerulein-induced ROS production, translocation of NADPH oxidase cytosolic subunits p 47(phox) and p 67(phox) to the membrane, and the expression of apoptotic genes and apoptotic indices, as compared to the cells without treatment and those transfected with the corresponding sense oligonucleotides. These results indicate that NADPH oxidase may mediate ROS-induced apoptosis in pancreatic acinar cells in a Ca(2+)-dependent manner.

Animals↗
PubMed2006

Noxa1 is a central component of the smooth muscle NADPH oxidase in mice.

NADPH oxidase is the most important source of oxygen-derived radicals (ROS) in the vascular wall. In vascular smooth muscle cells (VSMC), NADPH oxidase is characterized by the expression of the membrane subunit Nox1, which is activated by cytoplasmic proteins binding to its activation domain. We set out to identify the cytoplasmic protein involved in NADPH oxidase activation in mouse VSMC. Western blot analysis revealed that human endothelial cells and leukocytes but not VSMC from the aorta of the rat and the mouse express the classic NADPH oxidase activator p67phox. In mouse VSMC, however, the p67phox homologue Noxa1 was detected. Using antibodies generated against mouse Noxa1, the protein was observed in the cytosolic fraction of mouse VSMC with a molecular weight of about 51 kDa. Immunohistochemistry revealed that Noxa1 is expressed in the smooth muscle layer but not in endothelium or the adventitia of the mouse carotid artery. Fluorescent fusion proteins of Noxa1 were observed to be expressed in the cytoplasm of VSMC and coexpression of the NADPH oxidase organizer Noxo1 targeted the complex to membrane. An antisense plasmid of Noxa1 attenuated the endogenous Noxa1 protein expression in VSMC. This plasmid attenuated the ROS formation in mouse VSMC as detected using L012 chemiluminescence and prevented the agonist-induced ROS production in response to basic fibroblast growth factor and epidermal growth factor. In conclusion, these data indicate that Noxa1 replaces p67phox in VSMC and plays a central role in the activation of the NADPH oxidase in the vascular wall.

Adaptor Proteins, Signal Transducing↗
PubMed2005

NAD(P)H oxidase-stimulating activity of serum from type 2 diabetic patients with retinopathy mediates enhanced endothelial expression of E-selectin.

Endothelial expression of E-selectin is enhanced in diabetic patients with retinopathy, however, the underlying mechanisms are unclear. Therefore, this study was aimed to determine if endothelial expression of E-selectin is stimulated with serum from type 2 diabetic patients with retinopathy, and whether this process is related to NAD(P)H oxidase-derived oxidative stress. Serum was obtained from type 2 diabetic patients with (T2DR) or without (T2DM) retinopathy, and age-matched non-diabetic healthy person (Control). Serum was added to in vitro-grown human coronary artery endothelial cells (HCAEC), after which E-selectin expression, reactive oxygen species (ROS) production, and NAD(P)H oxidase activity were measured. Serum from T2DR induced a significantly higher expression of E-selectin than serum from T2DM and control in association with an enhanced production of ROS in HCAEC. T2DR serum enhanced E-selectin expression in a ROS-dependent manner since this process was significantly attenuated not only by tiron (1 mM), a superoxide scavenger, but also by DPI (10 micromol/L) and apocynin (100 micromol/L), inhibitors of NAD(P)H oxidase. Furthermore, the activity of NADH oxidase was markedly increased by T2DR serum, and this was accompanied by the enhanced membrane translocation of p47phox, a cytosolic subunit of NAD(P)H oxidase. These findings suggest that serum from T2DR induced up-regulation of E-selectin expression in HCAEC, and this process might be dependent on activation of endothelial NADH oxidase via an enhanced membrane translocation of p47phox.

1,2-Dihydroxybenzene-3,5-Disulfonic Acid Disodium ↗
PubMed2004

Expression and purification of his-tagged rat peroxisomal acyl-CoA oxidase I wild-type and E421 mutant proteins.

Rat peroxisomal acyl-CoA oxidase I is a key enzyme for the beta-oxidation of fatty acids, and the deficiency of this enzyme in patients has been previously reported. We cloned the gene of rat peroxisomal acyl-CoA oxidase I into a bacterial expression vector pLM1 with six continuous histidine codons attached to the 5' end of the gene. The cloned gene was overexpressed in Escherichia coli and the soluble protein was purified with a nickel HiTrap chelating metal-affinity column in 90% yield to apparent homogeneity. The specific activity of the purified His-tagged rat peroxisomal acyl-CoA oxidase I was 1.5 micromol/min/mg. It has been proposed that Glu421 is a catalytic residue responsible for deprotonation of alpha-proton of acyl-CoA substrate. We constructed four mutant expression plasmids of the enzyme, pACO(E421D), pACO(E421A), pACO(E421Q), and pACO(E421G) using site-directed mutagenesis. Mutant proteins were overexpressed in E. coli and purified with a nickel metal-affinity column. Kinetic studies of wild-type and mutant proteins were carried out, and the result confirmed that Glu421 is a catalytic residue of rat peroxisomal acyl-CoA oxidase I. Our overexpression in E. coli and one-step purification of the highly active N-terminal His-tagged rat peroxisomal acyl-CoA oxidase I greatly facilitated our further investigation of this enzyme, and our result from site-directed mutagenesis increased our understanding of the mechanism for the reaction catalyzed by peroxisomal acyl-CoA oxidase I.

Acyl-CoA Oxidase↗