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PubMed1995

Pre- and post-translational regulation of lysyl oxidase by transforming growth factor-beta 1 in osteoblastic MC3T3-E1 cells.

The final enzymatic step required for collagen cross-linking is the extracellular oxidative deamination of peptidyl-lysine and -hydroxylysine residues by lysyl oxidase. A cross-linked collagenous extracellular matrix is required for bone formation. The goals of this study were to compare the transforming growth factor (TGF)-beta 1 regulation of lysyl oxidase enzyme activity and steady state mRNA levels to changes in COL1A1 mRNA levels in MC3T3-E1 osteoblastic cells. TGF-beta 1 increased steady state lysyl oxidase and COL1A1 mRNA levels in a dose- and time-dependent manner. The increase in lysyl oxidase mRNA levels was transient, peaking at 12 h and 8.8 times controls in cells treated with 400 pM TGF-beta 1. COL1A1 steady state mRNA levels increased maximally to 3.5-fold of controls. Development of increased lysyl oxidase enzyme activity was delayed and was of slightly lower magnitude than the increase in its mRNA levels. This suggested limiting post-translational processing of lysyl oxidase proenzyme. Pulse-labeling/immunoprecipitation studies demonstrated slow proenzyme secretion and proteolytic processing. Development and application of an independent assay for lysyl oxidase proenzyme proteolytic processing activity verified its proportionately lower stimulation by 400 pM TGF-beta 1. Thus, lysyl oxidase regulation by TGF-beta 1 in osteoblastic cell cultures occurs at both pre- and post-translational levels. This regulation is consistent with increased production of a collagenous extracellular matrix.

3T3 Cells↗
PubMed1996

Regulation of lysyl oxidase by basic fibroblast growth factor in osteoblastic MC3T3-E1 cells.

Lysyl oxidase catalyzes the final known enzymatic step required for collagen and elastin cross-linking. A cross-linked collagenous extracellular matrix is required for bone formation. This study investigated whether lysyl oxidase, like its type I collagen substrate, is down-regulated by basic fibroblast growth factor (bFGF) in osteoblastic MC3T3-E1 cells and determined the degree of post-transcriptional control. Steady-state lysyl oxidase mRNA levels decreased to 30% of control after 24 h of treatment with 1 and 10 nm bFGF. This regulation was time-dependent. COL1A1 mRNA levels declined to less than 10% of control after 24 h of bFGF treatment. Media lysyl oxidase activity decreased consistent with steady-state mRNA changes in cultures that were refed after 24 h of growth factor treatment. Interestingly, treatment of MC3T3-E1 cells with 0.01-0.1 nm bFGF for 24 h and treatment with 1 nm bFGF for up to 12 h resulted in a modest stimulation of lysyl oxidase gene expression and enzyme activity. At least 50% of the down-regulation of lysyl oxidase was shown to be posttranscriptional. New protein synthesis was not required for the down-regulation by bFGF, but cycloheximide did increase constitutive lysyl oxidase mRNA levels 2.5-fold. We conclude that lysyl oxidase and COL1A1 are regulated similarly by bFGF in these osteoblastic cells, consistent with the in vivo effects of this growth factor on bone collagen metabolism.

3T3 Cells↗
PubMed1996

Cloning and characterization of the yeast HEM14 gene coding for protoporphyrinogen oxidase, the molecular target of diphenyl ether-type herbicides.

Protoporphyrinogen oxidase, which catalyzes the oxygen-dependent aromatization of protoporphyrinogen IX to protoporphyrin IX, is the molecular target of diphenyl ether type herbicides. The structural gene for the yeast protoporphyrinogen oxidase, HEM14, was isolated by functional complementation of a hem14-1 protoporphyrinogen oxidase-deficient yeast mutant, using a novel one-step colored screening procedure to identify heme-synthesizing cells. The hem14-1 mutation was genetically linked to URA3, a marker on chromosome V, and HEM14 was physically mapped on the right arm of this chromosome, between PRP22 and FAA2. Disruption of the HEM14 gene leads to protoporphyrinogen oxidase deficiency in vivo (heme deficiency and accumulation of heme precursors), and in vitro (lack of immunodetectable protein or enzyme activity). The HEM14 gene encodes a 539-amino acid protein (59,665 Da; pI 9.3) containing an ADP- beta alpha beta-binding fold similar to those of several other flavoproteins. Yeast protoporphyrinogen oxidase was somewhat similar to the HemY gene product of Bacillus subtilis and to the human and mouse protoporphyrinogen oxidases. Studies on protoporphyrinogen oxidase overexpressed in yeast and purified as wild-type enzyme showed that (i) the NH2-terminal mitochondrial targeting sequence of protoporphyrinogen oxidase is not cleaved during importation; (ii) the enzyme, as purified, had a typical flavin semiquinone absorption spectrum; and (iii) the enzyme was strongly inhibited by diphenyl ether-type herbicides and readily photolabeled by a diazoketone derivative of tritiated acifluorfen. The mutant allele hem14-1 contains two mutations, L422P and K424E, responsible for the inactive enzyme. Both mutations introduced independently in the wild-type HEM14 gene completely inactivated the protein when analyzed in an Escherichia coli expression system.

Aerobiosis↗
PubMed2000

Lysyl oxidase activates the transcription activity of human collagene III promoter. Possible involvement of Ku antigen.

Lysyl oxidase is an extracellular enzyme that controls the maturation of collagen and elastin. Lysyl oxidase and collagen III often show similar expression patterns in fibrotic tissues. Therefore, we investigated the influence of lysyl oxidase overexpression on the promoter activity of human COL3A1 gene. Our results showed that when COS-7 cells overexpressed the mature form of lysyl oxidase, the activity of the human COL3A1 promoter was increased up to an average of 12 times when tested by luciferase reporter assay. The effect was specific, because other promoters were not affected. Moreover, lysyl oxidase effect was abolished by beta-aminopropionitrile, a specific inhibitor of its catalytic activity. Electrophoretic mobility shift assay showed a binding activity in the region from -101 to -77 that was significantly increased by lysyl oxidase overexpression. The binding was specifically competed by the cold probe, and the mutagenesis of this region abolished both the binding activity in gel retardation and lysyl oxidase stimulation of COL3A1 promoter in transfection experiments. We identified the binding activity as Ku antigen in its two components: Ku80 and Ku70. This study suggests a new coordinated mechanism by which lysyl oxidase might control the development of fibrosis.

Aminopropionitrile↗
PubMed2001

Lysyl oxidase-like protein from bovine aorta. Isolation and maturation to an active form by bone morphogenetic protein-1.

Recently several cDNAs have been described encoding lysyl oxidase-like proteins. Their deduced amino acid sequences are characterized by a strong similarity in the C-terminal region, corresponding to the lysyl oxidase family catalytic domain, and by marked differences in the N-terminal regions. Different biological functions have been described for lysyl oxidases in addition to their traditionally assumed cross-linking role. To answer the question of whether these different functions are carried out by different lysyl oxidases, purified and active forms of these enzymes are required. At present only the classical form of lysyl oxidase has been purified and characterized. The purpose of this study was to isolate and characterize the lysyl oxidase-like protein. In view of the strong sequence homology with the C-terminal domain of other lysyl oxidases, we chose to purify the protein from bovine aorta using antibodies specific to the N-terminal domain of the proenzyme. We have isolated a 56-kDa protein identified by amino acid sequencing as the bovine lysyl oxidase-like precursor, which is cleaved at the Arg-Arg-Arg sequence at positions 89-91 by a furin-like activity, as revealed after deblocking of the N-terminal residue. The immunopurified protein was largely inactive, but further processing in vitro by bone morphogenetic protein-1 led to an enzyme that was active on elastin and collagen substrates.

Amino Acid Oxidoreductases↗
PubMed2002

NADPH oxidase-dependent oxidation and externalization of phosphatidylserine during apoptosis in Me2SO-differentiated HL-60 cells. Role in phagocytic clearance.

Resolution of inflammation requires clearance of activated neutrophils by phagocytes in a manner that protects adjacent tissues from injury. Mechanisms governing apoptosis and clearance of activated neutrophils from inflamed areas are still poorly understood. We used dimethylsulfoxide-differentiated HL-60 cells showing inducible oxidase activity to study NADPH oxidase-induced apoptosis pathways typical of neutrophils. Activation of the NADPH oxidase by phorbol myristate acetate caused oxidative stress as shown by production of superoxide and hydrogen peroxide, depletion of intracellular glutathione, and peroxidation of all three major classes of membrane phospholipids, phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine. In addition, phorbol myristate acetate stimulation of the NADPH oxidase caused apoptosis, as evidenced by apoptosis-specific phosphatidylserine externalization, increased caspase-3 activity, chromatin condensation, and nuclear fragmentation. Furthermore, phorbol myristate acetate stimulation of the NADPH oxidase caused recognition and ingestion of dimethylsulfoxide-differentiated HL-60 cells by J774A.1 macrophages. To reveal the apoptosis-related component of oxidative stress in the phorbol myristate acetate-induced response, we pretreated cells with a pancaspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (z-VAD-fmk), and found that it caused partial inhibition of hydrogen peroxide formation as well as selective protection of only phosphatidylserine, whereas more abundant phospholipids, phosphatidylcholine and phosphatidylethanolamine, were oxidized to the same extent in the absence or presence of z-VAD-fmk. In contrast, inhibitors of NADPH oxidase activity, diphenylene iodonium and staurosporine, as well as antioxidant enzymes, superoxide dismutase/catalase, completely protected all phospholipids against peroxidation, inhibited expression of apoptotic biomarkers and externalization of phosphatidylserine, and reduced phagocytosis of differentiated HL-60 cells by J774A.1 macrophages. Similarly, zymosan-induced activation of the NADPH oxidase resulted in the production of superoxide and oxidation of different classes of phospholipids of which only phosphatidylserine was protected by z-VAD-fmk. Accordingly, zymosan caused apoptosis in differentiated HL-60 cells, as evidenced by caspase-3 activation and phosphatidylserine externalization. Finally, zymosan triggered caspase-3 activation and extensive SOD/catalase-inhibitable phosphatidylserine exposure in human neutrophils. Overall, our results indicate that NADPH oxidase-induced oxidative stress in neutrophil-like cells triggers apoptosis and subsequent recognition and removal of these cells through pathways dependent on oxidation and externalization of phosphatidylserine.

Amino Acid Chloromethyl Ketones↗
PubMed2003

Cu(I)-dependent biogenesis of the galactose oxidase redox cofactor.

Galactose oxidase is a copper metalloenzyme containing a novel protein-derived redox cofactor in its active site, formed by cross-linking two residues, Cys228 and Tyr272. Previous studies have shown that formation of the tyrosyl-cysteine (Tyr-Cys) cofactor is a self-processing step requiring only copper and dioxygen. We have investigated the biogenesis of cofactor-containing galactose oxidase from pregalactose oxidase lacking the Tyr-Cys cross-link but having a fully processed N-terminal sequence, using both Cu(I) and Cu(II). Mature galactose oxidase forms rapidly following exposure of a pregalactose oxidase-Cu(I) complex to dioxygen (t(1/2) = 3.9s at pH7). In contrast, when Cu(II) is used in place of Cu(I) the maturation process requires several hours (t(1/2) = 5.1 h). EDTA prevents reaction of pregalactose oxidase with Cu(II) but does not interfere with the Cu(I)-dependent biogenesis reaction. The yield of cross-link corresponds to the amount of copper added, although a fraction of the pregalactose oxidase protein is unable to undergo this cross-linking reaction. The latter component, which may have an altered conformation, does not interfere with analysis of cofactor biogenesis at low copper loading. The biogenesis product has been quantitatively characterized, and mechanistic studies have been developed for the Cu(I)-dependent reaction, which forms oxidized, mature galactose oxidase and requires two molecules of O2. Transient kinetics studies of the biogenesis reaction have revealed a pH sensitivity that appears to reflect ionization of a protein group (pKa = 7.3) at intermediate pH resulting in a rate acceleration and protonation of an early oxygenated intermediate at lower pH competing with commitment to cofactor formation. These spectroscopic, kinetic, and biochemical results lead to new insights into the biogenesis mechanism.

Aspergillus niger↗
PubMed2004

Distinct ligand-dependent roles for p38 MAPK in priming and activation of the neutrophil NADPH oxidase.

In response to certain cytokines and inflammatory mediators, the activity of the neutrophil NADPH oxidase enzyme is primed for enhanced superoxide production when the cells receive a subsequent oxidase-activating stimulus. The relative role of p38 MAPK in the priming and activation processes is incompletely understood. We have developed a 2-step assay that allows the relative contributions of p38 MAPK activity in priming to be distinguished from those involved in oxidase activation. Using this assay, together with in vitro kinase assays and immunochemical studies, we report that p38 MAPK plays a critical role in TNFalpha priming of the human and porcine NADPH oxidase for superoxide production in response to complement-opsonized zymosan (OpZ), but little, if any, role in neutrophil priming by platelet-activating factor (PAF) for OpZ-dependent responses. The OpZ-mediated activation process per se is independent of p38 MAPK activity, in contrast to oxidase activation by fMLP, where 70% of the response is eliminated by p38 MAPK inhibitors regardless of the priming agent. We further report that incubation of neutrophils with TNFalpha results in the p38 MAPK-dependent phosphorylation of a subpopulation of p47(phox) and p67(phox) molecules, whereas PAF priming results in phosphorylation only of p67(phox). Despite these phosphorylations, TNFalpha priming does not result in significant association of either of these oxidase subunits with neutrophil membranes, demonstrating that the molecular basis for priming does not appear to involve preassembly of the NADPH oxidase holoenzyme/cytochrome complex prior to oxidase activation.

Animals↗
PubMed2006

Plant sulfite oxidase as novel producer of H2O2: combination of enzyme catalysis with a subsequent non-enzymatic reaction step.

Sulfite oxidase (EC 1.8.3.1) from the plant Arabidopsis thaliana is the smallest eukaryotic molybdenum enzyme consisting of a molybdenum cofactor-binding domain but lacking the heme domain that is known from vertebrate sulfite oxidase. While vertebrate sulfite oxidase is a mitochondrial enzyme with cytochrome c as the physiological electron acceptor, plant sulfite oxidase is localized in peroxisomes and does not react with cytochrome c. Here we describe results that identified oxygen as the terminal electron acceptor for plant sulfite oxidase and hydrogen peroxide as the product of this reaction in addition to sulfate. The latter finding might explain the peroxisomal localization of plant sulfite oxidase. 18O labeling experiments and the use of catalase provided evidence that plant sulfite oxidase combines its catalytic reaction with a subsequent non-enzymatic step where its reaction product hydrogen peroxide oxidizes another molecule of sulfite. In vitro, for each catalytic cycle plant SO will bring about the oxidation of two molecules of sulfite by one molecule of oxygen. In the plant, sulfite oxidase could be responsible for removing sulfite as a toxic metabolite, which might represent a means to protect the cell against excess of sulfite derived from SO2 gas in the atmosphere (acid rain) or during the decomposition of sulfur-containing amino acids. Finally we present a model for the metabolic interaction between sulfite and catalase in the peroxisome.

Acid Rain↗
PubMed2004

NADPH oxidase-mediated generation of reactive oxygen species is critically required for survival of undifferentiated human promyelocytic leukemia cell line HL-60.

Nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase) mediated generation of reactive oxygen species (ROS) was originally identified as the powerful host defense machinery against microorganism in phagocytes. But recent reports indicated that some non-phagocytic cells also have the NADPH oxidase activity, and the ROS produced by it may act as cell signal molecule. But as far as today, whether the NADPH oxidase also plays similar role in phagocyte has not been paid much attention. Utilizing the undifferentiated HL-60 promyelocytic leukemia cells as a model, the aim of the present study was to determine whether NADPH oxidase plays a role on ROS generation in undifferentiated HL-60, and the ROS mediated by it was essential for cell's survival. For the first time, we verified that the release of ROS in undifferentiated HL-60 was significantly increased by the stimulation with Calcium ionophore or opsonized zymosan, which are known to trigger respiration burst in phagocytes by NADPH oxidase pathway. Diphenylene iodonium (DPI) or apocynin (APO), two inhibitors of NADPH oxidase, significantly suppressed the increasing of ROS caused by opsonized zymosan. Cell survival assay and fluorescence double dyeing with acridine orange and ethidium bromide showed that DPI and APO, as well as superoxide dismutase (SOD) and catalase (CAT) concentration-dependently decreased the viability of undifferentiated HL-60 cells, whereas exogenous H2O2 can rescue the cells from death obviously. Our results suggested that the ROS, generated by NADPH oxidase play an essential role in the survival of undifferentiated HL-60 cells.

Acetophenones↗
PubMedDate not supplied

Phenylacetaldehyde oxidation by freshly prepared and cryopreserved guinea pig liver slices: the role of aldehyde oxidase.

Phenylacetaldehyde is formed when the xenobiotic and biogenic amine 2-phenylethylamine is inactivated by a monoamine oxidase-catalyzed oxidative deamination. Exogenous phenylacetaldehyde is found in certain foodstuffs such as honey, cheese, tomatoes, and wines. 2-Phenylethylamine can trigger migraine attacks in susceptible individuals and can become fairly toxic at high intakes from foods. It may also function as a potentiator that enhances the toxicity of histamine and tyramine. The present investigation examines the metabolism of phenylacetaldehyde to phenylacetic acid in freshly prepared and in cryopreserved guinea pig liver slices. In addition, it compares the relative contribution of aldehyde oxidase, xanthine oxidase, and aldehyde dehydrogenase in the oxidation of phenylacetaldehyde using specific inhibitors for each oxidizing enzyme. The inhibitors used were isovanillin for aldehyde oxidase, allopurinol for xanthine oxidase, and disulfiram for aldehyde dehydrogenase. In freshly prepared liver slices, phenylacetaldehyde was converted mainly to phenylacetic acid, with traces of 2-phenylethanol being present. Disulfiram inhibited phenylacetic acid formation by 80% to 85%, whereas isovanillin inhibited acid formation to a lesser extent (50% to 55%) and allopurinol had little or no effect. In cryopreserved liver slices, phenylacetic acid was also the main metabolite, whereas the 2-phenylethanol production was more pronounced than that in freshly prepared liver slices. Isovanillin inhibited phenylacetic acid formation by 85%, whereas disulfiram inhibited acid formation to a lesser extent (55% to 60%) and allopurinol had no effect. The results in this study have shown that, in freshly prepared and cryopreserved liver slices, phenylacetaldehyde is converted to phenylacetic acid by both aldehyde dehydrogenase and aldehyde oxidase, with no contribution from xanthine oxidase. Therefore, aldehyde dehydrogenase is not the only enzyme responsible in the metabolism of phenylacetaldehyde, but aldehyde oxidase may also be important and thus its role should not be ignored.

Acetaldehyde↗
PubMed2006

The phosphoinositide-binding protein p40phox activates the NADPH oxidase during FcgammaIIA receptor-induced phagocytosis.

Superoxide produced by the phagocyte reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is essential for host defense. Enzyme activation requires translocation of p67(phox), p47(phox), and Rac-GTP to flavocytochrome b558 in phagocyte membranes. To examine the regulation of phagocytosis-induced superoxide production, flavocytochrome b558, p47(phox), p67(phox), and the FcgammaIIA receptor were expressed from stable transgenes in COS7 cells. The resulting COS(phox)FcgammaR cells produce high levels of superoxide when stimulated with phorbol ester and efficiently ingest immunoglobulin (Ig)G-coated erythrocytes, but phagocytosis did not activate the NADPH oxidase. COS7 cells lack p40(phox), whose role in the NADPH oxidase is poorly understood. p40(phox) contains SH3 and phagocyte oxidase and Bem1p (PB1) domains that can mediate binding to p47(phox) and p67(phox), respectively, along with a PX domain that binds to phosphatidylinositol-3-phosphate (PI(3)P), which is generated in phagosomal membranes. Expression of p40(phox) was sufficient to activate superoxide production in COS(phox)FcgammaR phagosomes. FcgammaIIA-stimulated NADPH oxidase activity was abrogated by point mutations in p40(phox) that disrupt PI(3)P binding, or by simultaneous mutations in the SH3 and PB1 domains. Consistent with an essential role for PI(3)P in regulating the oxidase complex, phagosome NADPH oxidase activation in primary macrophages ingesting IgG-coated beads was inhibited by phosphatidylinositol 3 kinase inhibitors to a much greater extent than phagocytosis itself. Hence, this study identifies a role for p40(phox) and PI(3)P in coupling FcgammaR-mediated phagocytosis to activation of the NADPH oxidase.

Animals↗
PubMed1986

A diphenol oxidase gene is part of a cluster of genes involved in catecholamine metabolism and sclerotization in drosophila. I. Identification of the biochemical defect in Dox-A2 [l(2)37Bf] mutants.

Phenol oxidase, a complex enzyme, plays a major role in the processes of sclerotization and melanization of cuticle in insects. Several loci have been reported to affect levels of phenol oxidase activity, but to date only one structural locus has been identified [Dox-3F (2-53.1+)]. Recently isolated Dox-A2 mutations (2-53.9) are recessive, early larval lethals, which as heterozygotes reduce phenol oxidase activity. A homozygous mutant escaper had weak, completely unpigmented cuticle and unpigmented bristles. Enzyme assays show that Dox-A2 heterozygotes have diphenol oxidase activity reduced to 47-79% of wild type, whereas monophenol oxidase activity, at 94-106% of wild type, is normal. Elevated pool sizes of the diphenol oxidase substrates DOPA, dopamine, and N-acetyldopamine are observed in the mutant, confirming the enzyme assay results. Separation of the three phenol oxidase A component activities on polyacrylamide gels shows that Dox-A2 mutations reduce the activity of only the A2 component. Dox-A2 may identify a structural locus for the A2 component of the diphenol oxidase enzyme system. The Dox-A2 locus is one of 18 loci in the dopa decarboxylase, Df (2L)TW130 region of the second chromosome, at least 14 of which affect the formation, melanization or sclerotization of cuticle in some way. These loci form an apparent cluster of functionally related genes.

Alleles↗
PubMed2005

Stimulation of NADPH oxidase by angiotensin II in human neutrophils is mediated by ERK, p38 MAP-kinase and cytosolic phospholipase A2.

OBJECTIVE: The present research was designed to study the involvement of ERK and p38 MAP-kinase in cytosolic phospholipase A2 (cPLA2) and NADPH-oxidase activation by angiotensin II (Ang II) in human neutrophils. METHODS: NADPH-oxidase activity was measured by reduction of cytochrome C. cPLA2 activity was measured in cell lysate using sonicated dispersions of 1-stearoyl-2-[C]arachidonyl phosphatidylcholine. Cells were incubated with MEK inhibitor UO126 or with p38 MAP-kinase inhibitor SB202190 prior to stimulation with Ang II. Translocation of p47, p67 and cPLA2 and phosphorylation of ERK and p38 MAP-kinase were measured by immunoblot analysis. RESULTS: Ang II induced a dose-dependent activation of NADPH oxidase in neutrophils and monocytes as well as in differentiated PLB-985 cells towards neutrophil or monocyte lineages, but not in cPLA2-deficient differentiated PLB-985 cells. An immediate activation of both ERK and p38 MAP-kinase and of cPLA2 was induced by Ang II in human neutrophils. In addition, Ang II induced translocation of the cytosolic oxidase components, detected by translocation of p47, which preceded the translocation of cPLA2 induced by this agonist. The p38 MAP-kinase inhibitor SB202190 or the MEK-ERK pathway inhibitor UO126 totally inhibited the activation of both NADPH oxidase and cPLA2 as well as the translocation of cytosolic oxidase components and of cPLA2 to the membrane fractions. CONCLUSIONS: These results suggest that either ERK or p38 MAP-kinase are involved in the activation of both cPLA2 and NADPH oxidase, and that cPLA2 is required for activation of the NADPH oxidase by Ang II in human neutrophils.

Adult↗
PubMed1994

Urate oxidase is imported into peroxisomes recognizing the C-terminal SKL motif of proteins.

Rat liver urate oxidase synthesized from cDNA through coupled transcription and translation was incubated at 26 degrees C for 60 min with purified peroxisomes from rat liver. Urate oxidase was efficiently imported into the peroxisomes, as determined by resistance to externally added proteinase K. The amount of imported urate oxidase increased with time and the import was temperature dependent. A synthetic peptide composed of the C-terminal 10 amino acid residues of acyl-CoA oxidase (the C-terminal tripeptide is Ser-Lys-Leu) inhibited the import of urate oxidase, whereas other peptides, in which the C-terminal Ser-Lys-Leu (SKL) sequence was deleted or mutated, were not effective. Two mutant urate oxidase proteins in which the C-terminal Ser-Arg-Leu (SRL) sequence was deleted or mutated to Ser-Glu-Leu (SEL) were not imported into peroxisomes. With substitution of a lysine residue for arginine in the SRL tripeptide at the C-terminus the import activity was retained. These results show that urate oxidase is important into peroxisomes via a common pathway with acyl-CoA oxidase, and that the C-terminal SRL sequence functions as a peroxisomal-targeting signal.

Amino Acid Sequence↗
PubMed1995

Xanthine oxidase mediates paraquat-induced toxicity on cultured endothelial cell.

The role of xanthine oxidase in paraquat toxicity was investigated using cultured bovine pulmonary artery endothelial cells. Exposure to paraquat 0.1 mM was done for 24 hr with or without tungsten pretreatment and in the presence or absence of xanthine oxidase inhibitors. Exposure to paraquat significantly increased O2- production and relative xanthine oxidase activity (xanthine oxidase activity divided by total xanthine dehydrogenase plus xanthine oxidase) while depressing cell growth. In contrast, tungsten and allopurinol inhibited the increase of xanthine oxidase activity and decreased O2- release. Cell injury was assessed by leakage of lactate dehydrogenase and by fluorescein diacetate staining; it was found that oxidase inhibitors (both allopurinol and tungsten) reduced paraquat cytotoxicity. Thus the toxicity of paraquat was at least partly due to intracellular O2- production mediated by xanthine oxidase and the subsequent formation of other free radicals.

Allopurinol↗
PubMed2003

Xanthine oxidase-catalyzed metabolism of 2-nitrofluorene, a carcinogenic air pollutant, in rat skin.

The reductive metabolism of 2-nitrofluorene, a carcinogenic air pollutant, in rat skin microsomes and cytosol was investigated. 2-Nitrofluorene was reduced to the corresponding amine by the microsomes with NADPH and by the cytosol with 2-hydroxypyrimidine or 4-hydroxypyrimidine under anaerobic conditions. The cytosolic activity was much higher than that of skin microsomes. The 2- or 4-hydroxypyrimidine-linked nitroreductase activity was inhibited by oxypurinol and (+/-)-8-(3-methoxy-4-phenylsulfinylphenyl) pyrazolo[1,5-a]-1,3,5-triazine-4(1H)-one (BOF-4272), inhibitors of xanthine oxidase, but not by menadione, chlorpromazine and isovanillin, inhibitors of aldehyde oxidase. When skin cytosol was applied to a DEAE-cellulose column, the fractions containing xanthine oxidase exhibited a marked 2-hydroxypyrimidine-linked nitroreductase activity. In contrast, the aldehyde oxidase fraction showed little activity. Nitroreductase fractions obtained by ion exchange chromatography showed a band in Western blotting analysis using anti-rat xanthine oxidase. Moreover, the xanthine oxidase fraction exhibited a significant nitroreductase activity in the presence of 2-hydroxypyrimidine, 4-hydroxypyrimidine or hypoxanthine, and these activities were inhibited by inhibitors of xanthine oxidase. These results indicated that reduction of 2-nitrofluorene in the skin was mainly catalyzed by xanthine oxidase.

Air Pollutants↗
PubMed1998

Role of arachidonic acid and its metabolites in the priming of NADPH oxidase in human polymorphonuclear leukocytes by peritoneal dialysis effluent.

Peritoneal dialysis effluent (PDE) contains a low-molecular-weight solute that will activate and prime the NADPH oxidase of human neutrophils via a phospholipase A2 (PLA2)-dependent mechanism. Since the products of PLA2 are known to activate and prime the oxidase we have investigated their role in the dialysis effluent-mediated activation and priming of human neutrophils. NADPH oxidase activity of PDE-primed and -unprimed neutrophils was measured by lucigenin-enhanced chemiluminescence in the presence of known inhibitors of the arachidonic acid cascade. Incubation of neutrophils with the nonselective PLA2 inhibitor quinacrine (0 to 100 microM) reduced oxidase activity in both primed and unprimed cells. Furthermore, primed cells were more sensitive to the action of quinacrine than were unprimed cells. We were unable to determine the relative roles of secretory PLA2 (sPLA2) and cytosolic PLA2 (cPLA2) since the selective sPLA2 inhibitor scalaradial (0 to 100 microM) inhibited oxidase activity in both groups of cells by similar degrees, while the specific cPLA2 inhibitor AACO-CF3 (0 to 50 microM) failed to affect activity in either group. Inhibition of platelet-activating factor (PAF), cycloxygenase, and 5-lipoxygenase-activating protein by hexanolamino-PAF (0 to 25 microM), flurbiprofen (0 to 25 microM), and MK886 (0 to 5 microM), respectively, had no effect upon oxidase activity. However, the direct inhibition of 5-lipoxygenase by caffeic acid or lipoxin A4 resulted in a similar concentration-dependent attenuation of oxidase activity in both primed and unprimed cells. Leukotriene B4 (LTB4) release from primed neutrophils was comparable to that from unprimed cells with the exception of phorbol myristate acetate-stimulated cells, which released fivefold more LTB4 than control. Taken together, these results suggest that it is arachidonic acid per se, and not its metabolites, that is important in priming of the neutrophil NADPH oxidase by dialysis effluent.

Anti-Inflammatory Agents↗