Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “OXIDASES”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,729 records · Page 96Linked to original sources
PubMed1993

TRAMP (tyrosine rich acidic matrix protein), a protein that co-purifies with lysyl oxidase from porcine skin. Identification of TRAMP as the dermatan sulphate proteoglycan-associated 22K extracellular matrix protein.

A protein (M(r)24 K) that co-purifies with porcine skin lysyl oxidase (M(r)34 K) has been isolated and characterised. Five variants of the 24 K protein were identified by Mono Q ion-exchange FPLC, as were four variants of lysyl oxidase. Amino acid analysis and partial sequencing revealed near identity of a 36-residue CNBr peptide from porcine skin lysyl oxidase to corresponding regions of the putative lysyl oxidase precursor derived from rat and human cDNA. The 24 K protein was found to be unrelated to lysyl oxidase, but comparison with a protein sequence database showed it to be the same as a recently described protein from bovine skin that is associated with dermatan sulphate proteoglycans. The 24 K protein is relatively rich in tyrosine, and isoelectric focussing shows it to be acidic, with pI's in the range 4.1 to 4.4. In view of these properties, we propose the name TRAMP (Tyrosine Rich Acidic Matrix Protein) to identify this protein. Though TRAMP appears not to be glycosylated, several experiments indicate the presence of sulphotyrosine residues. When assayed using an elastin substrate, the activity of lysyl oxidase is unaffected by TRAMP.

Alcian Blue↗
PubMed1995

Induction of human monocyte motility by lysyl oxidase.

Lysyl oxidase highly purified from calf aorta was found to be a potent chemotactic agent for unstimulated human peripheral blood mononuclear cells, determined in in vitro assays in Boyden chambers. A typical chemotactic bell-shaped curve was observed, with a maximal migratory response of 237% of control occurring at 10(-10) M lysyl oxidase. The chemotactic response was prevented by prior heat inactivation of the enzyme, by treatment of the enzyme with beta-aminopropionitrile or ethylenediamine, which are active site-directed inhibitors of lysyl oxidase, and by a competing, lysine-containing peptide substrate of lysyl oxidase. The chemoattractant response to lysyl oxidases was characterized by both chemokinetic and chemotactic components. These results raise the possibility that extracellular lysyl oxidase may have important roles to play in biology in addition to its established function in the crosslinking of elastin and collagen.

Aminopropionitrile↗
PubMed2003

Modification of analytical procedures for determining vitamin C enzyme (L-gulonolactone oxidase) activity in swine liver.

Modifications of the analytical method to determine L-gulono-gamma-lactone oxidase (EC 1.1.8) enzyme activity were conducted in pig liver by evaluating the concentration of added substrate (L-gulono-gamma-lactone), glutathione, and various tissue sample-to-buffer ratios in the incubation mixture. Sampling different liver sites (lobes), the effect of different cooling temperatures of the liver immediately after collection, and the effect of tissue storage length on subsequent enzyme activity were evaluated. Our results demonstrated that 10 mM of substrate added to the reaction media maximized L-gulono-gamma-lactone oxidase enzyme activity, whereas increasing levels of glutathione did not greatly affect enzyme activity. High sample-to-buffer ratios resulted in higher L-gulono-gamma-lactone oxidase activities but sample analytical variations and background interferences were greater. A 1:4 tissue sample to buffer ratio (weight:weight) resulted in repeatable values, but the importance of maintaining the same ratio of the two components seems to be critical within an experiment. Expressing L-gulono-gamma-lactone oxidase enzyme activity on a liver protein rather than on a liver weight basis also resulted in more consistent results. No difference in liver L-gulono-gamma-lactone oxidase enzyme activities or ascorbic acid concentrations occurred between liver lobes. L-gulono-gamma-lactone oxidase enzyme activity from 0 to 90 day of storage was not affected when tissue samples were immediately frozen in liquid nitrogen, or placed on crushed ice. During a 90-day storage the oxidized form of ascorbic acid (dehydroascorbic acid) decreased (P < 0.01), the reduced (ascorbic acid) form increased (P < 0.01), while total ascorbic acid concentration remained constant.

Animals↗
PubMed2002

The length of cytochrome c oxidase-negative segments in muscle fibres in patients with mtDNA myopathy.

Heteroplasmic mitochondrial DNA mutations often cause a skeletal myopathy associated with a mosaic distribution of cytochrome c oxidase-deficient muscle fibres. The function of an individual muscle fibre is dependent upon the metabolic activity throughout its length, but little is known about the length of cytochrome c oxidase-deficient segments in human skeletal muscle in patients with mitochondrial disease. We studied cytochrome c oxidase activity by serial section analysis of quadriceps muscle from two patients. We observed a striking variation in the length of the cytochrome c oxidase-negative segments. The shortest segments were 10 microm long, and the longest segment was the entire length of the larger biopsy (> or =1.2 mm). The lengths of the cytochrome c oxidase-negative segments were generally shorter in the less severely affected biopsy, and we frequently observed non-contiguous segments of cytochrome c oxidase deficiency within the same muscle fibre. The findings have important implications for our understanding of the pathogenesis and progression of mitochondrial DNA myopathy.

Adult↗
PubMed1992

A substrate-cofactor free radical intermediate in the reaction mechanism of copper amine oxidase.

Reduction of copper amine oxidase with substrate led to the appearance of a free radical which can be detected in anaerobiosis by ESR and optical spectroscopy. The origin of this radical was examined through studies of the semiquinones of 6-hydroxydopamine, an analogue of the recently identified cofactor 6-hydroxydopa. The ESR spectrum of the 6-hydroxydopamine radical was too narrow to account for the enzyme radical signal; however, after spontaneous reaction with primary amines the hyperfine splittings and spectral width obtained by modulation broadening became very similar to those observed for the oxidase radical species. This effect was ascribed to covalent binding of a nitrogen atom directly to the aromatic ring structure, suggesting that the amine oxidase radical is an amino-6-hydroxydopa semiquinone. Identical ESR spectra were obtained using the amines putrescine, cadaverine, p-[(dimethylamino)methyl]benzylamine, and ethylenediamine; these oxidase substrates gave identical enzyme radical spectra as well. The interaction between cofactor and substrate was proved unambiguously by the technique of isotopic labeling: addition of [15N2]ethylenediamine instead of the normal 14N-labeled compound changed the ESR spectra of both the enzyme radical and its 6-hydroxydopamine counterpart. The results were confirmed by optical spectroscopy measurements; 6-hydroxydopamine and oxidized 6-hydroxydopamine gave spectra identical to those of reduced and oxidized amine oxidase, respectively. The 6-hydroxydopamine radical showed a sharp peak at 440 nm; upon addition of amines the maximum shifted to 460 nm, as found for the enzyme. It is proposed that copper amine oxidase represents the first example of a mixed substrate-cofactor radical within the family of tyrosine radical enzymes.

Amine Oxidase (Copper-Containing)↗
PubMed1994

p21rac does not participate in the early interaction between p47-phox and cytochrome b558 that leads to phagocyte NADPH oxidase activation in vitro.

The phagocyte superoxide-generating NADPH oxidase, a multicomponent, membrane-bound electron transport chain, consists of cytochrome b558, p47-phox, p67-phox, and p21rac1 or p21rac2. The mechanisms of oxidase assembly are poorly understood. In previous studies using a cell-free NADPH oxidase system, we showed that preincubation of neutrophil membrane with neutrophil cytosol containing p47-phox, but not p67-phox, led to formation of a long-lived NADPH oxidase intermediate. This suggested that p47-phox interacted with cytochrome b558 in the early stages of oxidase assembly while p67-phox participated in a later stage. Peptides containing the sequence RGVHFIF (corresponding to amino acids 559-565 of the 91-kDa subunit of cytochrome b558) inhibit NADPH oxidase activity by blocking the early interaction between p47-phox and cytochrome b558. In the present study, we examined whether p21rac facilitated the interaction between p47-phox and cytochrome b558. We preincubated pure recombinant p47-phox with neutrophil membrane containing cytochrome b558 in the cell-free system. Superoxide-generating activity was subsequently reconstituted by adding pure rp67-phox and partially purified p21rac. RGVHFIF inhibited superoxide production if added to the cell-free system during preincubation of rp47-phox with membrane. RGVHFIF was markedly less inhibitory if added to the cell-free system after membrane was preincubated with pure rp47-phox. In contrast to p47-phox, preincubation of membrane with either p21rac or rp67-phox conferred no protection from inhibition of superoxide-generating activity by RGVHFIF added after preincubation.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1991

Kinetic comparison of reduction and intramolecular electron transfer in milk xanthine oxidase and chicken liver xanthine dehydrogenase by laser flash photolysis.

A comparative study using laser flash photolysis of the kinetics of reduction and intramolecular electron transfer among the redox centers of chicken liver xanthine dehydrogenase and of bovine milk xanthine oxidase is described. The photogenerated reductant, 5-deazariboflavin semiquinone, reacts with the dehydrogenase (presumably at the Mo center) in a second-order manner, with a rate constant (k = 6 x 10(7) M-1 s-1) similar to that observed with the oxidase [k = 3 x 10(7) M-1 s-1; Bhattacharyya et al. (1983) Biochemistry 22, 5270-5279]. In the case of the dehydrogenase, neutral FAD radical formation is found to occur by intramolecular electron transfer (kobs = 1600 s-1), presumably from the Mo center, whereas with the oxidase the flavin radical forms via a bimolecular process involving direct reduction by the deazaflavin semiquinone (k = 2 x 10(8) M-1 s-1). Biphasic rates of Fe/S center reduction are observed with both enzymes, which are due to intramolecular electron transfer (kobs approximately 100 s-1 and kobs = 8-11 s-1). Intramolecular oxidation of the FAD radical in each enzyme occurs with a rate constant comparable to that of the rapid phase of Fe/S center reduction. The methylviologen radical, generated by the reaction of the oxidized viologen with 5-deazariboflavin semiquinone, reacts with both the dehydrogenase and the oxidase in a second-order manner (k = 7 x 10(5) M-1 s-1 and 4 x 10(6) M-1 s-1, respectively). Alkylation of the FAD centers results in substantial alterations in the kinetics of the reaction of the viologen radical with the oxidase but not with the dehydrogenase. These results suggest that the viologen radical reacts directly with the FAD center in the oxidase but not in the dehydrogenase, as is the case with the deazaflavin radical. The data support the conclusion that the environments of the FAD centers differ in the two enzymes, which is in accord with other studies addressing this problem from a different perspective [Massey et al. (1989) J. Biol. Chem. 264, 10567-10573]. In contrast, the rate constants for intramolecular electron transfer among the Mo, FAD, and Fe/S centers in the two enzymes (where they can be determined) are quite similar.

Animals↗
PubMed1988

Inhibition by heparin of the oxidation of lysine in collagen by lysyl oxidase.

The generation of covalent cross-linkages in collagen is initiated by the deamination by lysyl oxidase of specific lysine residues in this connective tissue protein. Since lysyl oxidase activity is influenced by ionic ligands bound to its protein substrates, the effect of heparin, an anionic glycosaminoglycan known to bind to collagen, was explored by using collagen and elastin substrates and highly purified lysyl oxidase. Concentrations of heparin up to 1 mg mL-1 had little effect on the enzymatic rate of oxidation if it was added prior to the addition of enzyme to a preformed fibrillar collagen substrate or to an insoluble elastin substrate. However, collagen oxidation was inhibited by 85% if this glycosaminoglycan was present at 0.4 mg mL-1 during collagen fibril formation before addition of the enzyme. Similarly, the rate and extent of collagen fibrillogenesis in the absence of lysyl oxidase were each markedly inhibited in the presence of 0.4 mg mL-1 heparin. Heparin also inhibited the extent of tight binding of lysyl oxidase to preformed fibrils by about 40% under conditions where enzyme activity against preformed fibrils was hardly affected. These results suggest that heparin may modulate the oxidation and thus the insolubilization of extracellular collagen fibers, possibly under conditions where elastin fiber synthesis is not affected, and that the tight binding of lysyl oxidase to collagen is not completely related to the expression of enzyme activity toward this substrate. These results also have mechanistic implications for the retarding effect of heparin on postoperative wound healing.

Amino Acid Oxidoreductases↗
PubMed2004

Phosphorylated p40PHOX as a negative regulator of NADPH oxidase.

The leukocyte NADPH oxidase catalyzes the production of O(2)(-) from oxygen at the expense of NADPH. Activation of the enzyme requires interaction of the cytosolic factors p47(PHOX), p67(PHOX), and Rac2 with the membrane-associated cytochrome b(558). Activation of the oxidase in a semirecombinant cell-free system in the absence of an amphiphilic activator can be achieved by phosphorylation of the cytosolic factor p47(PHOX) by protein kinase C. Another cytosolic factor, p40(PHOX), was recently shown to be phosphorylated on serine and threonine residues upon activation of NADPH oxidase, but both stimulatory and inhibitory roles were reported. In the present study, we demonstrate that the addition of phosphorylated p40(PHOX) to the cell-free system inhibits NADPH oxidase activated by protein kinase C-phosphorylated p47(PHOX), an effect not observed with the unphosphorylated p40(PHOX). Moreover phosphorylated p40(PHOX) inhibits the oxidase if added before or after full activation of the enzyme. Direct mutagenesis of protein kinase C consensus sites enables us to conclude that phosphorylation of threonine 154 is required for the inhibitory effect of p40(PHOX) to occur. Although the phosphorylated mutants and nonphosphorylated mutants are still able to interact with both p47(PHOX) and p67(PHOX) in pull-down assays, their proteolysis pattern upon thrombin treatment suggests a difference in conformation between the phosphorylated and nonphosphorylated mutants. We postulate that phosphorylation of p40(PHOX) on threonine 154 leads to an inhibitory conformation that shifts the balance toward an inhibitory role and blocks oxidase activation.

Alanine↗
PubMed1997

Kinetic mechanism and cofactor content of soybean root nodule urate oxidase.

The kinetic mechanism of urate oxidase isolated from soybean root nodules has been determined by initial velocity kinetic studies monitoring oxygen uptake, in order to avoid potential artifacts in the spectrophotometric assay which arise from absorbance due to unidentified products of the enzymatic reaction. Urate and O2 bind to the enzyme sequentially; xanthine is a competitive inhibitor versus urate and a noncompetitive inhibitor versus O2, which suggests that urate binds to the enzyme before O2. This kinetic mechanism was confirmed by an 18O isotope-trapping experiment, which demonstrated that O2 does not bind productively to the enzyme in the absence of urate. The pH dependence of V and (V/K)urate reveal the presence of an ionizable residue on the enzyme with a pK of approximately 6.2, which must be unprotonated for the catalytic reaction to occur. The (V/K)O2 profile is pH independent; these data are accomodated by a model in which a unimolecular step intervenes between the binding of urate and O2. The pKi profile for 9-methylurate, a competitive inhibitor versus urate, is pH independent, confirming that the protonation state of the ionizable residue is not important for binding. The pKi profile for xanthine defines a pK of 7.4, which demonstrates that the monoanion of xanthine binds to the enzyme; by analogy, the monoanion of urate is predicted to be the substrate. The four isomeric N-methylurates were examined as potential inhibitors of urate oxidase. Only 9-methylurate showed significant inhibition suggesting that ionization at N9 of urate is not required for binding; it is proposed that the N3-deprotonated urate monoanion is the species which binds to urate oxidase. The gene encoding urate oxidase was cloned from soybeans and expressed in Escherichia coli. The metal content of the recombinant enzyme was examined by inductively coupled argon plasma emission spectroscopy, and only trace quantities of copper were found. The molecular mass of the protein was determined by MALDI-TOF mass spectrometry and found to be 35,059.8 Da. The calculated molecular mass of urate oxidase is 35,052 Da; therefore, these data suggest that there is no covalently bound cofactor in urate oxidase.

Cloning, Molecular↗
PubMed1997

Activation of the leukocyte NADPH oxidase subunit p47phox by protein kinase C. A phosphorylation-dependent change in the conformation of the C-terminal end of p47phox.

The leukocyte NADPH oxidase of neutrophils is a membrane-bound enzyme that catalyzes the production of O2- from oxygen using NADPH as electron donor. Dormant in resting neutrophils, the enzyme acquires catalytic activity when the cells are exposed to appropriate stimuli. During activation, the cytosolic oxidase components p47phox and p67phox migrate to the plasma membrane, where they associate with cytochrome b558, a membrane-bound flavohemoprotein, to assemble the active oxidase. An essential element of the activation process is the phosphorylation of p47phox, an event that accompanies oxidase activation in whole cells and can activate the oxidase in a cell-free system. We show here that the phosphorylation of p47phox leads to a substantial decrease in the reactivity of cysteine C378 toward N-ethylmaleimide, indicating the occurrence of a conformational change involving the C-terminal region of p47phox. A similar conformational change occurs when p47phox is exposed to arachidonate, one of a number of anionic detergents that activate the oxidase in the cell-free system. We propose that this change in conformation results in the appearance of a binding site through which p47phox interacts with cytochrome b558 during the activation process.

Amino Acid Sequence↗
PubMed2004

Inhibition of apple polyphenol oxidase activity by procyanidins and polyphenol oxidation products.

The rate of consumption of dissolved oxygen by apple polyphenol oxidase in cider apple juices did not correlate with polyphenol oxidase activity in the fruits and decreased faster than could be explained by the decrease of its polyphenolic substrates. The kinetics parameters of a crude polyphenol oxidase extract, prepared from apple (Braeburn cultivar), were determined using caffeoylquinic acid as a substrate. Three apple procyanidin fractions of n 80, 10.5, and 4 were purified from the parenchyma of cider apples of various cultivars. Procyanidins, caffeoylquinic acid, (-)-epicatechin, and a mixture of caffeoylquinic acid and (-)-epicatechin were oxidized by reaction with caffeoylquinic acid o-quinone in order to form oxidation products. All the fractions were evaluated for their inhibitory effect on PPO activity. Native procyanidins inhibited polyphenol oxidase activity, the inhibition intensity increasing with n. The polyphenol oxidase activity decreased by 50% for 0.026 g/L of the fraction of n 80, 0.17 g/L of the fraction of n 10.5, and 1 g/L of the fraction of n 4. The inhibitory effect of oxidized procyanidins was twice that of native procyanidins. Oxidation products of caffeoylquinic acid and (-)-epicatechin also inhibited polyphenol oxidase.

Beverages↗
PubMed2003

Two pathways of activation of the superoxide-generating NADPH oxidase of phagocytes in vitro--distinctive effects of inhibitors.

The NADPH oxidase complex of phagocytes comprises a membrane-associated flavocytochrome b559, and 4 cytosolic components: p47phox, p67phox, p40phox, and the small GTPase Rac. Activation of the oxidase in vivo is the result of assembly of the cytosolic components with cytochrome b559 and is mimicked in vitro by a cell-free system consisting of membranes, p47phox, p67phox, nonprenylated or prenylated Rac, and an anionic amphiphile as activator (defined as "p47phox and amphiphile-dependent" or canonical pathway). We reported that prenylated Rac1 is capable of activating the NADPH oxidase in vitro in the absence of p47phox and amphiphile (defined as "p47phox and amphiphile-independent" pathway). We now demonstrate that the 2 pathways exhibit distinctive susceptibilities to inhibitors: 1) The anionic amphiphile lithium dodecyl sulfate, an activator of the canonical pathway, has the opposite effect (inhibition) on oxidase activation by prenylated Rac and p67phox; 2) GDP and, paradoxically, GTP (but not GMP, ATP, ADP, and AMP) prevent oxidase activation by the p47phox and amphiphile-independent pathway but do not affect activation by the canonical pathway; 3) The Rac-binding domain of p21-activated kinase is a potent inhibitor of activation by the p47phox and amphiphile-independent pathway while exerting a milder inhibitory effect on the canonical pathway; 4) The C-terminal polybasic Rac1 peptide 177-191 and the cationic antibiotic neomycin sulfate inhibit activation by the canonical pathway but do not affect activation by the p47phox and amphiphile-independent pathway; 5) Binding of prenylated Rac1 to membrane-mimicking phospholipid vesicles is, nevertheless, enhanced when these contain negatively charged lipids. It is proposed that preferential inhibition of oxidase activation, via the p47phox and amphiphile-independent pathway, is a reflection of interference by the inhibitors with Rac-dependent recruitment of p67phox to the membrane.

Animals↗
PubMed1991

Use of caffeine metabolite ratios to explore CYP1A2 and xanthine oxidase activities.

Caffeine was used as a metabolic probe to screen healthy subjects for their activities of two enzymes, deduced to be CYP1A2 (an inducible cytochrome P450) and xanthine oxidase. A longitudinal study revealed modest effects of caffeine dose, ethanol intake, and time-of-day on the CYP1A2 index, without any effect on the xanthine oxidase index. The coefficients of intraindividual variation not accounted for were 5.0% for the xanthine oxidase and 17.2% for the CYP1A2 index. In a population study, both indexes showed a log normal distribution, with CYP1A2 values of most subjects covering a 6.3-fold range but only a 1.7-fold range with xanthine oxidase. The CYP1A2 index was 33% decreased in women who used oral contraceptives and substantially increased in cigarette smokers. Neither the CYP1A2 nor the xanthine oxidase index differed between volunteers of Chinese and European extraction. Four of 178 subjects showed unexplained low xanthine oxidase values (i.e., values several standard deviations below the mean).

Adult↗
PubMed1992

Racial and gender differences in N-acetyltransferase, xanthine oxidase, and CYP1A2 activities.

BACKGROUND: The urinary molar concentration ratios of several caffeine metabolites are indicators of specific drug metabolizing enzyme activities. The ratios of 5-acetyl-amino-6-formylamino-3-methyluracil (AFMU) to 1-methylxanthine (1X), AFMU to 1X plus 1-methyluric acid (1U), and AFMU to 1X + 1U + AFMU are indicative of N-acetyltransferase (NAT) activity; the ratios of 1U to 1X and 1U to 1U + 1X indicate xanthine oxidase activity; and the ratio of the sum of 7-demethylated metabolites (AFMU + 1X + 1U) to the precursor for all three compounds, paraxanthine (PX), is a putative indicator of CYP1A2 oxidative activity. Our objective was to discern whether there are race-, gender-, and age-related differences in these indexes of drug-metabolizing activity. METHODS: In 342 normal healthy unrelated subjects, metabolites were measured in urine collected after administration of low-dose caffeine. RESULTS: By two-way analysis of variance, NAT activity was higher in black subjects than in white subjects when assessed as AFMU/(1U + 1X) or as AFMU/(AFMU + 1U + 1X) (p = 0.001 and p = 0.002, respectively), but less so by use of AFMU/1X (p = 0.08). Xanthine oxidase activity, as assessed by 1U/1X or as 1U/(1U + 1X), was lower in black subjects than in white subjects (p = 0.02 and p = 0.001, respectively) and was lower in males than in females (p = 0.001 for both ratios). Females had higher AFMU/1X ratios (p = 0.03) because of higher xanthine oxidase activity. In a model in which AFMU/1X was the dependent variable and race, sex, age, and an index of xanthine oxidase (1U/1X) were independent variables, only race and 1U/1X were significant determinants of this NAT index (p = 0.003 and p < 0.001, respectively). The CYP1A2 ratio was lower in black subjects (p = 0.036) and in females (p = 0.015). CONCLUSION: Racial and gender differences in xanthine oxidase activity render the AFMU/1X ratio less reliable as an assessment of NAT activity in a heterogeneous population compared with the AFMU/(1U + 1X) or AFMU/(AFMU + 1U + 1X) ratios. The observed racial and gender differences in NAT, xanthine oxidase, and CYP1A2 activities may have implications for racial and gender differences in drug effects and carcinogen biotransformation.

Adolescent↗
PubMed2003

The voltage dependence of NADPH oxidase reveals why phagocytes need proton channels.

The enzyme NADPH oxidase in phagocytes is important in the body's defence against microbes: it produces superoxide anions (O2-, precursors to bactericidal reactive oxygen species). Electrons move from intracellular NADPH, across a chain comprising FAD (flavin adenine dinucleotide) and two haems, to reduce extracellular O2 to O2-. NADPH oxidase is electrogenic, generating electron current (I(e)) that is measurable under voltage-clamp conditions. Here we report the complete current-voltage relationship of NADPH oxidase, the first such measurement of a plasma membrane electron transporter. We find that I(e) is voltage-independent from -100 mV to >0 mV, but is steeply inhibited by further depolarization, and is abolished at about +190 mV. It was proposed that H+ efflux mediated by voltage-gated proton channels compensates I(e), because Zn2+ and Cd2+ inhibit both H+ currents and O2- production. Here we show that COS-7 cells transfected with four NADPH oxidase components, but lacking H+ channels, produce O2- in the presence of Zn2+ concentrations that inhibit O2- production in neutrophils and eosinophils. Zn2+ does not inhibit NADPH oxidase directly, but through effects on H+ channels. H+ channels optimize NADPH oxidase function by preventing membrane depolarization to inhibitory voltages.

Animals↗
PubMed1999

'Outside-in' signalling mechanisms underlying CD11b/CD18-mediated NADPH oxidase activation in human adherent blood eosinophils.

1 Incubation of human eosinophils in BSA-coated tissue culture plates resulted in time-dependent adhesion and attendant activation of the NADPH oxidase that were both inhibited (by >85%) by blocking antibodies raised against CD11b and CD18. 2 SB 203580, an inhibitor of p38 mitogen-activated protein (MAP) kinase, did not influence adhesion but inhibited superoxide anion generation (pIC50=-6.57). 3 PP1, an inhibitor of the src-family of protein tyrosine kinases, inhibited adhesion and CD11b/CD18-mediated superoxide anion generation with similar potencies (pEC50s=-5.53 and -5.99 respectively) suggesting that inhibition of the NADPH oxidase was a direct consequence of blocking adhesion. 4 The protein kinase C (PKC) inhibitors Ro-31 8220 (broad spectrum inhibitor), GF 109203X (inhibitor of conventional and novel isoforms) and Gö 6976 (inhibitor of conventional isoforms) suppressed adhesion-dependent NADPH oxidase activation (pIC50s=-6.61, -6.05 and -4.89 respectively) without affecting adhesion. Based upon the selectivity of these drugs PKCdelta and PKCepsilon are implicated in the suppression of oxidant production. 5 Wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PtdIns 3-kinase), abolished superoxide anion production in adherent eosinophils (pEC50=-9.06). Similarly, CD11b/CD18-dependent adhesion was suppressed with the same potency (pEC50=-9.29) although the maximum effect did not exceed 50% implying that wortmannin also had an affect on those processes that govern adhesion-driven oxidase activation. 6 PD 098059 and piceatannol, inhibitors of MAP kinase kinase-1 and the syk tyrosine kinase respectively, had no effect on CD11b/CD18-mediated adhesion or NADPH oxidase activation. 7 The results of this study demonstrate that human eosinophils adhere to BSA-coated plastic by a CD11b/CD18-dependent mechanism, which is responsible for activation of the NADPH oxidase. Although the signalling pathway(s) utilized by CD11b/CD18 is still to be elucidated, the data presented herein implicate p38 MAP kinase, novel PKCs and PtdIns 3-kinase.

Androstadienes↗
PubMed2006

Flow-induced cerebral vasodilatation in vivo involves activation of phosphatidylinositol-3 kinase, NADPH-oxidase, and nitric oxide synthase.

Reactive oxygen species (ROS) such as superoxide (O2*-) and hydrogen peroxide (H2O2) are known cerebral vasodilators. A major source of vascular ROS is the flavin-containing enzyme nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase. Activation of NADPH-oxidase leads to dilatation of the basilar artery in vivo via production of H2O2, but the endogenous stimuli for this unique vasodilator mechanism are unknown. Shear stress is known to activate both NADPH-oxidase and phosphatidylinositol-3 kinase (PI3-K) in cultured cells. Hence, this study used a cranial window preparation in anesthetized rats to investigate whether increased intraluminal blood flow could induce cerebral vasodilatation via the activation of NADPH-oxidase and/or PI3-K. Bilateral occlusion of the common carotid arteries to increase basilar artery blood flow caused reproducible, reversible vasodilatation. Topical treatment of the basilar artery with the NADPH-oxidase inhibitor diphenyleneiodonium (DPI) (0.5 and 5 micromol/L) inhibited flow-induced dilatation by up to 50% without affecting dilator responses to acetylcholine. Treatment with the H2O2 scavenger, catalase similarly attenuated flow-induced dilatation, suggesting a role for NADPH-oxidase-derived H2O2 in this response. The nitric oxide synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) partially reduced flow-induced dilatation, and combined treatment with a ROS inhibitor (DPI or catalase) and L-NAME caused a greater reduction in flow-induced dilatation than that seen with any of these inhibitors alone. Flow-induced dilatation was also markedly inhibited by the PI3-K inhibitor, wortmannin. Increased O2*- production in the endothelium of the basilar artery during acute increases in blood flow was confirmed using dihydroethidium. Thus, flow-induced cerebral vasodilatation in vivo involves production of ROS and nitric oxide, and is dependent on PI3-K activation.

Animals↗