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PubMed2006

Activation of the superoxide-producing phagocyte NADPH oxidase requires co-operation between the tandem SH3 domains of p47phox in recognition of a polyproline type II helix and an adjacent alpha-helix of p22phox.

Activation of the superoxide-producing phagocyte NADPH oxidase, crucial for host defence, requires an SH3 (Src homology 3)-domain-mediated interaction of the regulatory protein p47phox with p22phox, a subunit of the oxidase catalytic core flavocytochrome b558. Although previous analysis of a crystal structure has demonstrated that the tandem SH3 domains of p47phox sandwich a short PRR (proline-rich region) of p22phox (amino acids 151-160), containing a polyproline II helix, it has remained unknown whether this model is indeed functional in activation of the oxidase. In the present paper we show that the co-operativity between the two SH3 domains of p47phox, as expected from the model, is required for oxidase activation. Deletion of the linker between the p47phox SH3 domains results not only in a defective binding to p22phox but also in a loss of the activity to support superoxide production. The present analysis using alanine-scanning mutagenesis identifies Pro152, Pro156 and Arg158 in the p22phox PRR as residues indispensable for the interaction with p47phox. Pro152 and Pro156 are recognized by the N-terminal SH3 domain, whereas Arg158 contacts with the C-terminal SH3 domain. Amino acid substitution for any of the three residues in the p22phox PRR abrogates the superoxide-producing activity of the oxidase reconstituted in intact cells. The bis-SH3-mediated interaction of p47phox with p22phox thus functions to activate the phagocyte oxidase. Furthermore, we provide evidence that a region C-terminal to the PRR of p22phox (amino acids 161-164), adopting an a-helical conformation, participates in full activation of the phagocyte oxidase by fortifying the association with the p47phox SH3 domains.

Amino Acid Substitution↗

PubMed1982

The dissociation of glucose oxidase by sodium n-dodecyl sulphate.

1. The enzymic activity of glucose oxidase was determined as a function of pH and sodium n-dodecyl sulphate (SDS) concentration. 2. Glucose oxidase is not deactivated by SDS at pH 6 even after prolonged incubation, but is deactivated at pH 4.3 and 3.65. 3. Sedimentation-rate analysis showed that glucose oxidase dissociates into its two subunits at pH 5 and below, and sedimentation-equilibrium experiments in the presence of SDS gave a subunit molecular weight of 73,500. 4. SDS binds to glucose oxidase in acid solutions; specific binding occurs ap pH 3.65, but at pH 6 only co-operative binding was observed. 5. Glucose oxidases in which some of the carboxy groups were blocked with glycine methyl ester were deactivated by SDS at pH 6.0; the rate of deactivation increased with the extent of esterification. 6. Deactivation of esterified glucose oxidases correlated with thermal analysis of the initial SDS interaction, the exothermicity of the interaction increasing with the extent of esterification. 7. The results show that carboxy groups confer resistance to deactivation by SDS on glucose oxidase by screening cationic residues and inhibiting specific interactions that facilitate dissociation into subunits.

Amino Acids↗

PubMed1986

Reactivity of a functional carbonyl moiety in bovine aortic lysyl oxidase. Evidence against pyridoxal 5'-phosphate.

Previous studies have pointed towards a cofactor role for pyridoxal 5'-phosphate (PLP) in lysyl oxidase, the enzyme that generates the peptidyl aldehyde precursor to the lysine-derived cross-linkages in elastin and collagen. The nature of a carbonyl moiety in purified bovine aortic lysyl oxidase was explored in the present study. A PLP dinitrophenylhydrazone could not be isolated from lysyl oxidase, although corresponding preparations of aspartate aminotransferase, a PLP-dependent enzyme, yielded this derivative, as revealed by h.p.l.c. Analysis of lysyl oxidase for PLP after reduction of the enzyme by NaBH4, a procedure that converts PLP-protein aldimines into stable 5'-phosphopyridoxyl functions, also proved negative in tests using monoclonal antibody specific for this epitope. Lysyl oxidase was competitively inhibited by phenylhydrazine, and inhibition became irreversible with time at 37 degrees C, displaying a first-order inactivation rate constant of 0.4 min-1 and KI of 1 microM. [14C]Phenylhydrazine was covalently incorporated into the enzyme in a manner that was prevented by prior modification of the enzyme with beta-aminopropionitrile, a specific active-site inhibitor, and which correlated with functional active-site content. The chemical stability of the enzyme-bound phenylhydrazine exceeded that expected of linkages between PLP and proteins. The absorption spectrum of the phenylhydrazine derivative of lysyl oxidase was clearly distinct from that of the phenylhydrazone of PLP. It is concluded that lysyl oxidase contains a carbonyl cofactor that is not identical with PLP and that is bound to the enzyme by a stable chemical bond.

Amino Acid Oxidoreductases↗

PubMed1987

Inhibition of peroxisomal fatty acyl-CoA oxidase by antimycin A.

Peroxisomal fatty acyl-CoA oxidase was inhibited by micromolar concentrations of antimycin A, an inhibitor of mitochondrial respiration. The inhibition was observed with all three substrates tested, i.e. palmitoyl-CoA, trihydroxycoprostanoyl-CoA and hexadecanedioyl-CoA. The peroxisomal D-amino acid oxidase was also inhibited by antimycin, but the peroxisomal L-alpha-hydroxyacid oxidase and uric acid oxidase and the mitochondrial monoamine oxidase were not. The degree of inhibition of acyl-CoA oxidase by antimycin was strongly dependent on the amount of cellular protein present in the assay mixture: at a fixed antimycin concentration, the inhibition was gradually lost with increasing protein concentrations. At a fixed cellular protein concentration in the assay mixtures, the mitochondrial oxidation of glutamate or palmitoylcarnitine was inhibited at antimycin concentrations that were much lower than those required for the inhibition of fatty acyl-CoA oxidase. Our results, nevertheless, demonstrate that antimycin A must be used with caution, when it is added to homogenates or subcellular fractions in order to distinguish between mitochondrial and peroxisomal fatty acid oxidation.

Acyl-CoA Oxidase↗

PubMed1989

D-aspartate oxidase in rat, bovine and sheep kidney cortex is localized in peroxisomes.

D-Aspartate oxidase (EC 1.4.3.1) was assayed in subcellular fractions and in highly purified peroxisomes from rat, bovine and sheep kidney cortex as well as from rat liver. During all steps of subcellular-fractionation procedures, D-aspartate oxidase co-fractionated with peroxisomal marker enzymes. In highly purified preparations of peroxisomes, the enrichment of D-aspartate oxidase activity over the homogenate is about 32-fold, being comparable with that of the peroxisomal marker enzymes catalase and D-amino acid oxidase. Disruption of the peroxisomes by freezing and thawing released more than 90% of the enzyme activity, which is typical for soluble peroxisomal-matrix proteins. Our findings provide strong evidence that in these tissues D-aspartate oxidase is a peroxisomal-matrix protein and should be added as an additional flavoprotein oxidase to the known set of peroxisomal oxidases.

Amino Acid Oxidoreductases↗

PubMed1998

Tumour necrosis factor alpha activates a p22phox-based NADH oxidase in vascular smooth muscle.

Increasing experimental evidence suggests that non-phagocytic cells express a potent superoxide (O2-.)-producing NADH oxidase that might be related to the phagocytic NADPH oxidase. Here we show that the cytokine tumour necrosis factor alpha (TNF-alpha) activates, in a time- and dose-dependent manner, a O2-.-producing NADH oxidase in cultured rat aortic smooth-muscle cells. Dose-response experiments for NADH showed an upward shift of the curve for TNF-alpha-treated cells, suggesting that TNF-alpha increased the amount of available enzyme. Using the anti-sense transfection technique, we further demonstrate that the molecular identity of this oxidase includes p22(phox) (the alpha subunit of cytochrome b558 and part of the electron transfer component of the phagocytic NADPH oxidase), which we recently cloned from a rat vascular smooth-muscle cell cDNA library. In addition, prolonged treatment with TNF-alpha increased p22phox mRNA expression without affecting p22phox mRNA stability, and only when transcriptional activity was intact. These findings identify a p22phox-containing NADH oxidase as a source for cytokine-induced free radical production in vascular smooth-muscle cells and clarify some of the mechanisms involved in the regulation of vascular oxidase activity.

Animals↗

PubMed2007

The role of PI3Ks in the regulation of the neutrophil NADPH oxidase.

The NADPH oxidase complex of neutrophils and macrophages is an important weapon used by these cells to kill microbial pathogens. The regulation of this enzyme complex is necessarily complicated by the diverse receptor types that are needed to trigger its activation and also the tight control that is required to deliver this activation at the appropriate time and place. As such, several signalling pathways have been established to regulate the NADPH oxidase downstream of cell surface receptors. Central amongst these are PI3K- (phosphoinositide 3-kinase)-dependent pathways, blockade of which severely limits activation of the oxidase to several soluble and particulate stimuli. The precise roles of the phosphoinositide products of PI3K activity in regulating NADPH oxidase assembly and activation are still unclear, but there is emerging evidence that they play a key role via regulation of guanine nucleotide exchange on Rac, a key component in the oxidase complex. There is also very strong evidence that the PI3K products PtdIns(3,4)P2 and PtdIns3P can bind directly to the PX (Phox homology) domains of the core oxidase components p47phox and p40phox respectively. However, the significance of these interactions in terms of membrane localization or allosteric consequences for the oxidase complex remains to be established.

Animals↗

PubMed1990

Effect of dietary protein restriction on renal purines and purine-metabolizing enzymes in adriamycin nephrosis in rats: a mechanism for protection against acute proteinuria involving xanthine oxidase inhibition.

1. A low protein diet prevents the development of proteinuria and glomerular damage in adriamycin experimental nephrosis without affecting renal haemodynamics. In this study the hypothesis was tested as to whether protein restriction is able to modulate the purine metabolic cycle and related enzymes such as xanthine oxidase, one of the putative effectors of adriamycin nephrotoxicity. 2. Renal activities of xanthine oxidase and purine nucleoside phosphorylase were markedly depressed in adriamycin-treated rats fed a 9% casein (low protein) diet compared with the group fed a 22% casein (normal protein) diet both 1 day after adriamycin administration and at the time of appearance of heavy proteinuria (day 15), whereas the activity of renal adenosine deaminase was unchanged. 3. The concentrations of the metabolic substrates of xanthine oxidase, i.e. hypoxanthine and xanthine, were constantly lower in renal homogenates of rats fed a low protein diet compared with those on a normal protein diet. In urine, uric acid, the product of hypoxanthine-xanthine transformation, was lower 1 day after adriamycin injection in protein-restricted rats compared with the group on a normal protein diet which showed a marked increase in its excretion. At the same time, the urinary efflux of adenosine 5'-monophosphate, which is the precursor nucleotide of the above-mentioned nucleosides and bases, was very high in rats fed a low protein diet, whereas it was absent in the group on a normal protein diet. 4. The progressive increment in proteinuria of glomerular origin (i.e. increased excretion of albumin and transferrin) typical of adriamycin-treated rats fed a normal protein diet was inhibited in the protein-restricted animals, which were normoproteinuric on day 10 and were only slightly proteinuric on day 15. 5. Like protein restriction, the pharmacological suppression of renal xanthine oxidase by dietary tungstate and the scavenging by dimethylthiourea of the putative free radical deriving from the action of xanthine oxidase, were associated with a similar (quantitative and qualitative) inhibition of glomerular proteinuria. 6. These data demonstrate that dietary protein restriction is associated with a block in purine metabolism within the kidney due to a marked reduction in the activities of two main enzymes of the cycle, i.e. purine nucleoside phosphorylase and xanthine oxidase, the latter being a putative effector of adriamycin nephrotoxicity. The partial reduction of proteinuria induced by a low protein diet is quantitatively and qualitatively comparable with the reduction induced by the specific block of renal xanthine oxidase or by the scavenging of OH.deriving from hypoxanthine and xanthine transformation.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed2003

Histamine inhibits neutrophil NADPH oxidase activity triggered by the lipoxin A4 receptor-specific peptide agonist Trp-Lys-Tyr-Met-Val-Met.

The vasoactive amine histamine is found at high concentrations in the immune and inflammatory tissues. Earlier studies have revealed that histamine regulates the nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase-dependent formation of oxygen radicals by phagocytic cells. However, the effects of histamine on intracellular signal transduction mechanisms of relevance to oxidase regulation remain controversial. For this study, we investigated the effects of histamine on NADPH oxidase activity in human neutrophil granulocytes triggered by a lipoxin A4 receptor agonist [the hexapeptide Trp-Lys-Tyr-Met-Val-Met (WKYMVM), a formyl peptide receptor (FPR) agonist (the chemotactic tripeptide formylmethionyl-leucyl-phenylalanine (fMLF)) and an activator of protein kinase C (phorbol myristate acetate (PMA)]. We report that histamine, acting via H2-type histamine receptors (H2R), suppresses NADPH oxidase-dependent formation of oxygen radicals induced by WKYMVM and fMLF but not that induced by PMA. Peptide-induced mobilization of granule-localized complement receptor 3 (CR3) was unaffected by histamine suggesting that the inhibition specifically affected NADPH oxidase activation. Our data suggest that histamine downregulates FPRL1- and FPR-induced NADPH oxidase activity upstream of protein kinase C (PKC) and downstream of the separation of the peptide-induced signal into granule secretion and oxidase activation.

Chemotactic Factors↗

PubMed1998

Glucose oxidase from Penicillium amagasakiense. Primary structure and comparison with other glucose-methanol-choline (GMC) oxidoreductases.

The complete amino acid sequence of glucose oxidase from Penicillium amagasakiense was determined by Edman degradation and mass spectrometry of peptide fragments derived from three different specific proteolytic digests and a cyanogen bromide cleavage. The complete sequence of each monomer comprises 587 amino acid residues, contains three cysteine residues, and seven potential N-glycosylation sites, of which at least five were confirmed to be glycosylated. Glucose oxidase from P. amagasakiense shows a high degree of identity (66%) and 79% similarity to glucose oxidase from Aspergillus niger, and is a member of the glucose-methanol-choline (GMC) oxidoreductase family. The tertiary structures of glucose oxidase from A. niger and cholesterol oxidase from Brevibacterium sterolicum were superimposed to provide a template for the sequence comparison of members of the GMC family. The general topology of the GMC oxidoreductases is conserved, with the exception of the presence of an active site lid in cholesterol oxidase and the insertion of additional structural elements in the substrate-binding domain of alcohol oxidase. The overall structure can be divided into five distinct sequence regions: FAD-binding domain, extended FAD-binding domain, flavin attachment loop and intermediate region, FAD covering lid, and substrate-binding domain. The FAD-binding and the extended FAD-binding domains are composed of several separate sequence regions. The other three regions each comprise a single contiguous sequence. Four major consensus patterns have been identified, including the nucleotide-binding consensus sequence close to their N-termini. The functions of the two motifs recently selected by the Genetics Computer Group, Madison, Wisconsin, as additional signature patterns of the GMC oxidoreductases are discussed. The other consensus patterns belong to either the FAD-binding or the extended FAD-binding domain. In addition, the roles of conserved residues are discussed wherever possible.

Amino Acid Sequence↗

PubMed1994

L-3,4-dihydroxyphenylalanine-induced dopamine release in the striatum of intact and 6-hydroxydopamine-treated rats: differential effects of monoamine oxidase A and B inhibitors.

Administration of L-DOPA (50 mg/kg) elicits a significant increase in extracellular dopamine in striata of rats treated with the catecholaminergic neurotoxin 6-hydroxydopamine but not in striata of intact rats. To assess the role of dopaminergic nerve terminals in determining the effects of exogenous L-DOPA on extracellular dopamine levels in striatum, we examined the relative contributions of monoamine oxidase A and monoamine oxidase B to the catabolism of dopamine synthesized from exogenous L-DOPA. Extracellular concentrations of dopamine and its catabolite, 3,4-dihydroxyphenylacetic acid, were monitored with in vivo dialysis in striata of intact rats and of rats with unilateral 6-hydroxydopamine lesions of striatal dopamine. Clorgyline (2 mg/kg), an inhibitor of monoamine oxidase A, significantly increased dopamine and decreased 3,4-dihydroxyphenylacetic acid in intact but not in dopamine-depleted striata. Inhibition of monoamine oxidase B with either L-deprenyl (1 mg/kg) or Ro 19-6327 (1 mg/kg) did not significantly affect dopamine or 3,4-dihydroxyphenylacetic acid in striata of intact or dopamine-depleted rats. In intact rats, administration of clorgyline in conjunction with L-DOPA produced a > 20-fold increase in dopamine and prevented the L-DOPA-induced increase in 3,4-dihydroxyphenylacetic acid. Although both L-deprenyl and Ro 19-6327 administered in combination with L-DOPA elicited a small but significant increase in dopamine, levels of 3,4-dihydroxyphenylacetic acid were not affected. In rats pretreated with 6-hydroxydopamine, clorgyline had no significant effect on the increases in dopamine and 3,4-dihydroxyphenylacetic acid elicited by L-DOPA. Furthermore, neither L-deprenyl nor Ro 19-6327 affected L-DOPA-induced increases in dopamine and 3,4-dihydroxyphenylacetic acid in dopamine-depleted striata. The present findings indicate that deamination by monoamine oxidase A is the primary mechanism for catabolism of striatal dopamine, both under basal conditions and after administration of exogenous L-DOPA. Loss of dopaminergic terminals eliminates this action of monoamine oxidase A but does not enhance deamination by monoamine oxidase B. These data support a model in which exogenous L-DOPA elicits enhanced extracellular accumulation of dopamine in the dopamine-depleted striatum because some transmitter synthesis occurs at nondopaminergic sites and the dopamine terminals that normally take up and catabolize this pool of transmitter are absent.

3,4-Dihydroxyphenylacetic Acid↗

PubMed2003

Modulation of p47PHOX activity by site-specific phosphorylation: Akt-dependent activation of the NADPH oxidase.

The leukocyte NADPH oxidase catalyzes the reduction of oxygen to O(2)(-) at the expense of NADPH. Extensive phosphorylation of the oxidase subunit p47(PHOX) occurs during the activation of the enzyme in intact cells. p47(PHOX) carrying certain serine-to-alanine mutations fails to support NADPH oxidase activity in intact cells, suggesting that the phosphorylation of specific serines on p47(PHOX) is required for the activation of the oxidase. Earlier studies with both intact cells and a kinase-dependent, cell-free system have suggested that protein kinase C can phosphorylate those serines of p47(PHOX) whose phosphorylation is necessary for its activity. Work with inhibitors suggested that a phosphatidylinositol 3-kinase-dependent pathway also can activate the oxidase. Phosphorylation of p47(PHOX) by Akt (protein kinase B), whose activation depends on phosphatidylinositol 3-kinase, could be the final step in such a pathway. We now find that Akt activates the oxidase in vitro by phosphorylating serines S304 and S328 of p47(PHOX). These results suggest that Akt could participate in the activation of the leukocyte NADPH oxidase.

Base Sequence↗

PubMed1971

Demonstration of imino acids as products of the reactions catalyzed by D- and L-amino acid oxidases.

It had long been thought, but never demonstrated, that imino acids are formed in the reactions catalyzed by D- and L-amino acid oxidases (EC 1.4.3.3 and 1.4.3.2). The formation of imino acids is now shown directly by allowing the amino acid oxidase reaction to proceed in the presence of NaBH(4), when the imino acid is reduced to the corresponding racemic amino acid. Thus, when NaBH(4) is added to a mixture of D-amino acid oxidase and D-alanine, a significant amount of L-alanine is formed. Analogous results are obtained using L-amino acid oxidase and L-leucine. Since D-amino acid oxidase is active in the presence of NaBH(4), L-alanine continues to be formed until most of the D-isomer is oxidized by the enzyme. This reaction provides a new method for inverting the configuration of an amino acid. When NaBH(4) is added to a system containing D-amino acid oxidase plus D-alanine and L-lysine, free epsilon-N-(1-carboxyethyl)-L-lysine is formed. When bovine serum albumin is substituted for L-lysine, the same compound results upon acid hydrolysis. It is concluded that the amino acid oxidase reaction produces a free imino acid, which may be reduced by NaBH(4) to a racemic amino acid or may form Schiff's bases by reaction with the epsilon-amino groups of proteins and of free lysine.

Alanine↗

PubMed1973

Molecular basis of the biological function of molybdenum: the relationship between sulfite oxidase and the acute toxicity of bisulfite and SO2.

The administration of tungsten to rats maintained on a low molybdenum diet resulted in a dose- and time-dependent loss of sulfite oxidase (EC 1.8.3.1) and xanthine oxidase (EC 1.2.3.2) activities and hepatic molybdenum. These tungsten-treated animals appeared healthy, but were more susceptible to bisulfite toxicity. The median lethal dose for intraperitoneal bisulfite was found to be 181 mg of NaHSO(3) per kg for the animals deficient in sulfite oxidase, compared to 473 mg/kg for normal rats. The survival time of rats exposed to SO(2) at concentrations of 590 ppm and higher was seen to be inversely related to the level of SO(2). At 590 ppm and 925 ppm, control animals displayed symptoms of severe respiratory toxicity before death. At 2350 ppm of SO(2), death was preceded by seizures and prostration, symptoms observed with the systemic toxicity of injected bisulfite. At 590 ppm, animals deficient in sulfite oxidase were indistinguishable from control animals. However, at 925 ppm and 2350 ppm, the deficient animals displayed symptoms of systemic toxicity and had much shorter survival times. It is concluded that sulfite oxidase is instrumental in counteracting the toxic systemic effects of bisulfite, either injected or derived from respired SO(2). Respiratory death probably results from the toxicity of gaseous SO(2) before absorption as bisulfite and cannot be alleviated by sulfite oxidase. Sulfite oxidase does not appear to be inducible by either bisulfite or SO(2).

Animals↗

PubMed1989

Cytosolic components of the respiratory burst oxidase: resolution of four components, two of which are missing in complementing types of chronic granulomatous disease.

The respiratory burst oxidase of neutrophils can be activated in a cell-free system in which plasma membranes, cytosol, Mg2+, and a membrane-perturbing detergent, such as arachidonate or sodium dodecyl sulfate, are all required. Using the technique of preparative isoelectric focusing, the cytosol factor required for oxidase activation was resolved into four components termed C1-C4 with respective pI values of approximately 3.1, 6.0, 7.0, and 9.5. Individually, these components were incapable of activating the oxidase and could only be detected in the presence of suboptimal amounts of normal cytosol that served to supply at least a limited amount of each of the required components. Attempts to activate the oxidase with a combination of the four components failed, suggesting that there might be a yet undetected fifth cytosolic component. Patients with autosomal recessive cytochrome b-positive chronic granulomatous disease (type II CGD) are severely deficient in cytosol factor activity. When added to cytosol samples from two patients with this form of CGD, component C4 restored the ability of each patient's cytosol to activate dormant oxidase. None of the other three cytosol factor components (C1-C3) was effective in this regard, a finding supported by the direct demonstration that these three components were present in normal amounts in this type of CGD. A different form of type II CGD was identified in a third patient on the basis of complementation studies in which the patient's cytosol was able to activate the oxidase in the cell-free system when mixed with cytosol from one of the first two patients. The defect in this third patient's cytosol could be partially corrected by component C2, but not component C4, obtained from normal cytosol. These findings indicate that the role of cytosol in the activation of the respiratory burst oxidase is more complex than previously appreciated in that at least four cytosolic components appear to be required. Defects in two of these components have now been identified and appear to be responsible for two biochemically distinct forms of CGD.

Adolescent↗

PubMed1996

PR-39, a proline-rich antibacterial peptide that inhibits phagocyte NADPH oxidase activity by binding to Src homology 3 domains of p47 phox.

Reactive oxygen intermediates generated by the phagocyte NADPH oxidase are critically important components of host defense. However, these highly toxic oxidants can cause significant tissue injury during inflammation; thus, it is essential that their generation and inactivation are tightly regulated. We show here that an endogenous proline-arginine (PR)-rich antibacterial peptide, PR-39, inhibits NADPH oxidase activity by blocking assembly of this enzyme through interactions with Src homology 3 domains of a cytosolic component. This neutrophil-derived peptide inhibited oxygen-dependent microbicidal activity of neutrophils in whole cells and in a cell-free assay of NADPH oxidase. Both oxidase inhibitory and direct antimicrobial activities were defined within the amino-terminal 26 residues of PR-39. Oxidase inhibition was attributed to binding of PR-39 to the p47phox cytosolic oxidase component. Its effects involve both a polybasic amino-terminal segment and a proline-rich core region of PR-39 that binds to the p47phox Src homology 3 domains and, thereby, inhibits interaction with the small subunit of cytochrome b558, p22phox. These findings suggest that PR-39, which has been shown to be involved in tissue repair processes, is a multifunctional peptide that can regulate NADPH oxidase production of superoxide anion O2-. thus limiting excessive tissue damage during inflammation.

Amino Acid Sequence↗

PubMed1996

p22phox is a critical component of the superoxide-generating NADH/NADPH oxidase system and regulates angiotensin II-induced hypertrophy in vascular smooth muscle cells.

Superoxide anion formation is vital to the microbicidal activity of phagocytes. Recently, however, there is accumulating evidence that it is also involved in cell growth in vascular smooth muscle cells (VSMCs). We have shown that the hypertrophic agent angiotensin II stimulates superoxide production by activating the membrane-bound NADH/NADPH oxidase and that inhibition of this oxidase attenuates vascular hypertrophy. However, the molecular identity of this oxidase in VSMCs is unknown. We have recently cloned the cytochrome b558 alpha-subunit, p22(phox) (one of the key electron transfer elements of the NADPH oxidase in phagocytes), from a rat VSMC cDNA library, but its role in VSMC oxidase activity remains unclarified. Here we report that the complete inhibition of p22(phox) mRNA expression by stable transfection of antisense p22(phox) cDNA into VSMCs results in a decrease in cytochrome b content, which is accompanied by a significant inhibition of angiotensin II-stimulated NADH/NADPH-dependent superoxide production, subsequent hydrogen peroxide production, and [3H]leucine incorporation. We provide the first evidence that p22(phox) is a critical component of superoxide-generating vascular NADH/NADPH oxidase and suggest a central role for this oxidase system in vascular hypertrophy.

Angiotensin II↗

PubMed1997

Cloning and functional expression of a mammalian gene for a peroxisomal sarcosine oxidase.

Sarcosine oxidation in mammals occurs via a mitochondrial dehydrogenase closely linked to the electron transport chain. An additional H2O2-producing sarcosine oxidase has now been purified from rabbit kidney. A corresponding cDNA was cloned from rabbit liver and the gene designated sox. This rabbit sox gene encodes a protein of 390 amino acids and a molecular mass of 44 kDa identical to the molecular mass estimated for the purified enzyme. Sequence analysis revealed an N-terminal ADP-betaalphabeta-binding fold, a motif highly conserved in tightly bound flavoproteins, and a C-terminal peroxisomal targeting signal 1. Sarcosine oxidase from rabbit liver exhibits high sequence homology (25-28% identity) to monomeric bacterial sarcosine oxidases. Both purified sarcosine oxidase and a recombinant fusion protein synthesized in Escherichia coli contain a covalently bound flavin, metabolize sarcosine, L-pipecolic acid, and L-proline, and cross-react with antibodies raised against L-pipecolic acid oxidase from monkey liver. Subcellular fractionation demonstrated that sarcosine oxidase is a peroxisomal enzyme in rabbit kidney. Transfection of human fibroblast cell lines and CV-1 cells (monkey kidney epithelial cells) with the sox cDNA resulted in a peroxisomal localization of sarcosine oxidase and revealed that the import into the peroxisomes is mediated by the peroxisomal targeting signal 1 pathway.

Amino Acid Sequence↗