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PubMed1998

Immunohistochemical localization of xanthine oxidase in human retina.

Xanthine oxidase has been established as an important source of oxygen free radicals in ischemia-reperfusion injury. It has been localized in many different tissues such as heart and intestine, but it has not yet been localized in the eye. Xanthine oxidase was detected using immunohistochemistry on paraformaldehyde/glutaraldehyde fixed cryosections. Antibodies used included rabbit antibovine xanthine oxidase antibody and rabbit antihuman xanthine oxidase antibody. Xanthine oxidase was detected in the capillary endothelium cells of blood vessels in the retina of bovine and post mortem human eyes. Whole mount preparation of human retinas showed xanthine oxidase present throughout the small capillary network. Furthermore, whole mounts showed that xanthine oxidase was present in cones. This was confirmed by using mouse anticalbindin antibody for co-labelling. It is possible that xanthine oxidase can be a source of oxidative damage in the retina following ischemia-reperfusion injury.

Animals↗

PubMed1998

Role of xanthine oxidase in hydrogen peroxide production.

Increased production of oxygen free radicals may play a role in many diseases such as hypertension. As evidence indicates that xanthine oxidase may be involved in creating these reactive oxygen species, experiments were performed to additionally characterize hydrogen peroxide (H2O2) production in xanthine oxidase catalyzed reactions. In vitro measurements of hydrogen peroxide production from the xanthine/xanthine oxidase reaction were performed in buffered saline using an electrochemical technique, and the effect of allopurinol on inhibition of xanthine oxidase was determined. Experiments were also performed in blood plasma to characterize endogenous hydrogen peroxide producing capability and xanthine oxidase activity. In the presence of sodium azide, an inhibitor of catalase, peroxide production was measured in plasma after adding xanthine or xanthine oxidase and the results were similar to those obtained in buffered saline. When only sodium azide was added to plasma, hydrogen peroxide was produced at a level of 36.1 +/- 7.6 microM (n = 5). From these measurements, endogenous xanthine oxidase activity was estimated to be 6.5 +/- 0.3 mU/ml (n = 5). These results suggests that sufficient substrate exists in plasma to produce micromolar levels of hydrogen peroxide and xanthine oxidase may catalyze these reactions.

Allopurinol↗

PubMed1985

Purification and properties of an O2-.-generating oxidase from bovine polymorphonuclear neutrophils.

A membrane-associated O2-.-generating oxidase has been purified from activated bovine polymorphonuclear neutrophils (PMN). The oxidase was extracted with Triton X-100 from a PMN membrane fraction largely devoid of lysosomal granules. The Triton extract was purified by a series of steps, including ion-exchange chromatography on DE-52 cellulose, gel filtration on Sephadex G-200, and isoelectric focusing. The O2-.-generating oxidase activity was assayed as a superoxide dismutase inhibitable cytochrome c reductase. The activity of the purified enzyme was strictly dependent on NADPH as electron donor. The purification factor with respect to the phorbol myristate acetate activated PMN was 75, and the recovery was about 6%. The reactivity of the purified oxidase was increased by 3-4-fold after incubation with asolectin. The minimum molecular weight of the oxidase, deduced from migration in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was 65 000 +/- 3000. The optimum pH of the oxidase was 7.5, its KM,NADPH was congruent to 30 microM, and its isoelectric point was at pH 5.0. The enzyme was inhibited by low concentrations of mersalyl (half-inhibition congruent to 10 microM) and Cibacron Blue (half-inhibition less than 10 microM). It was insensitive to 1 mM cyanide. Rapid loss of activity occurred at 0-2 degrees C, concomitantly with a decrease in sensitivity to superoxide dismutase: both activity and sensitivity to superoxide dismutase could be restored by addition of asolectin. The purified oxidase contained no spectrophotometrically detectable cytochrome b, and enzymatic assay failed to detect FAD in oxidase preparations subjected to heat treatment or trypsin digestion.

Animals↗

PubMed2003

Remarkable stabilization of neutrophil NADPH oxidase using RacQ61L and a p67phox-p47phox fusion protein.

Activation of the phagocyte NADPH oxidase occurs via assembly of cytosolic p47(phox), p67(phox), and Rac with the membrane-bound flavocytochrome b(558). Recently, we have found that p67(phox)-(1-210) (p67N) fused with p47(phox)-(1-286) (p47N) or with Rac efficiently stabilizes the oxidase in a cell-free reconstitution system. In an attempt to further stabilize the oxidase, we herein used a constitutively active Rac, RacQ61L, and examined its effect on the oxidase stability. The half-life (t(1/2)) of the activity reconstituted with wild-type Rac was 12 min at 37 degrees C, which was extended 6-fold by RacQ61L. Also, the stability of the oxidase without p47(phox) increased 8-fold using RacQ61L. RacQ61L had a higher affinity for the complex than wild-type Rac and increased the affinity of p67N for the complex. Far-western blotting showed an enhanced binding between RacQ61L and p67N. The oxidase was stabilized by nanomolar FAD, and RacQ61L lowered the FAD concentration required. The combination of RacQ61L and a fusion protein consisting of p67N and p47N produced an extremely stable enzyme (t(1/2) = 184 min at 37 degrees C). The effectiveness of RacQ61L and fusion proteins on stabilization was in the following order: p67N-Rac < p67N + RacQ61L < or = p67N-RacQ61L << p67N-p47N + RacQ61L. These results indicate that a tightly bound ternary complex of p67(phox), Rac, and p47(phox) is very effective in maintaining the oxidase and confirm that the longevity of the activated state requires continuous association of these components. This simple and efficient method of stabilization may provide a useful tool to elucidate the nature of the activated oxidase.

Enzyme Activation↗

PubMed1997

Limited proteolysis and X-ray crystallography reveal the origin of substrate specificity and of the rate-limiting product release during oxidation of D-amino acids catalyzed by mammalian D-amino acid oxidase.

Limited proteolysis of D-amino acid oxidase holoenzyme with trypsin cleaves the protein at Arg 221 and near the C-terminus, producing stable 25, 13.4, and 2 kDa polypeptides [Torri-Tarelli, G., Vanoni, M. A., Negri, A., & Curti, B. (1990) J. Biol. Chem. 265, 21242-21246]. The 25 and 13.4 kDa polypeptides remain associated to form a nicked D-amino acid oxidase species. This nicked protein form maintains the ability to bind FAD, but exhibits altered catalytic efficiency toward the oxidation of various D-amino acids when compared to native DAAO. Changes in substrate specificity were first monitored by measuring the activity in the presence of different amino acid substrates at various times during proteolysis. Three amino acid substrates were then selected for further analysis of the properties of the nicked D-amino acid oxidase species produced by limited tryptic proteolysis: D-serine, D-arginine, and D-alanine. The three D-amino acids represented limiting cases of the observed changes of enzyme activity on nicking: loss of activity, increase of activity, and minor activity changes, respectively. D-serine was found to be no longer a substrate of D-amino acid oxidase. D-arginine exhibited a 2.5-fold increased apparent maximum velocity although its Km value increased 2-fold with the nicked enzyme in comparison to the native species. D-alanine was oxidized 1.5-fold faster by the nicked D-amino acid oxidase at infinite substrate concentration, and its Km value increased approximately 4-fold. The Kd for benzoate, which was determined kinetically with D-alanine as the enzyme substrate, increased 17-fold in the nicked species. Primary deuterium kinetic isotope effects on V and V/K during the oxidation of D-alanine were also measured. (D)V/K increased from 1.4 +/- 0.2 to 1.8 +/- 0.3 on nicking, while (D)V increased from 1.04 +/- 0.1 to 2.53 +/- 0.5. All the observed changes of the values of the kinetic parameters and of the observed isotope effects are consistent with the hypothesis that nicking of D-amino acid oxidase at position 221 decreases the strength of binding of both substrates and products to the enzyme active site. The information obtained by limited tryptic proteolysis nicely complements that gathered from the analysis of the three-dimensional structure of D-amino acid oxidase in complex with benzoate, which was recently determined [Mattevi, A., Vanoni, M. A., Todone, F., Rizzi, M., Teplyakov, A., Coda, A., Bolognesi, M., & Curti, B. (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 7496-7501]. Arginine 221 is part of the 216-228 loop that covers the active site and contributes residues to substrate binding and catalysis. The limited proteolysis data support the hypothesis that this loop acts as a lid on the active site and controls both substrate specificity and the rate of turnover of D-amino acid oxidase.

Alanine↗

PubMed1998

Mutational analysis of novel effector domains in Rac1 involved in the activation of nicotinamide adenine dinucleotide phosphate (reduced) oxidase.

The small molecular weight GTP-binding protein Rac (1 or 2) is an obligatory participant in the activation of the superoxide-generating NADPH oxidase. Active NADPH oxidase can be reconstituted in a cell-free system, consisting of phagocyte-derived membranes, containing cytochrome b559, and the recombinant cytosolic proteins p47-phox, p67-phox, and Rac, supplemented with an anionic amphiphile as an activator. The cell-free system was used before for the analysis of structural requirements of individual components participating in the assembly of NADPH oxidase. In earlier work, we mapped four previously unidentified domains in Rac1, encompassing residues 73-81 (a), 103-107 (b), 123-133 (c), and 163-169 (d), as important for cell-free NADPH oxidase activation. The domains were defined by assessing the activation inhibitory effect of a series of overlapping peptides, spanning the entire length of Rac1 [Joseph, G., and Pick, E. (1995) J. Biol. Chem. 270, 29079-29082]. We now used the construction of Rac1/H-Ras chimeras, domain deletion, and point mutations, to ascertain the functional relevance of three domains (b, c, and d) predicted by "peptide walking" and to determine the importance of specific residues within these domains. This methodology firmly establishes the involvement of domains b and d in the activation of NADPH oxidase by Rac1 and identifies H103 and K166, respectively, as residues critical for the effector function of these two domains. The functional significance of domain c (insert region) could not be confirmed, as shown by the minor effect of deleting this domain on NADPH oxidase activation. Analysis of the three-dimensional structure of Rac1 reveals that residues H103 and K166 are exposed on the surface of the molecule. Modeling of the activity-impairing point mutations suggests that the effect on the ability to activate NADPH oxidase depends on the side chains of the mutated amino acids and not on changes in the global structure of the protein. In conclusion, we demonstrate the existence of two novel effector sites in Rac1, necessary for supporting NADPH oxidase activation, supplementing the canonical N-terminal effector region.

Amino Acid Substitution↗

PubMed1999

Kinetic study of the activation of the neutrophil NADPH oxidase by arachidonic acid. Antagonistic effects of arachidonic acid and phenylarsine oxide.

The O(2)(-) generating NADPH oxidase complex of neutrophils comprises two sets of components, namely a membrane-bound heterodimeric flavocytochrome b which contains the redox centers of the oxidase and water-soluble proteins of cytosolic origin which act as activating factors of the flavocytochrome. The NADPH oxidase can be activated in a cell-free system consisting of plasma membranes and cytosol from resting neutrophils in the presence of GTPgammaS and arachidonic acid. NADPH oxidase activation is inhibited by phenylarsine oxide (PAO), a sulfhydryl reagent for vicinal or proximal thiol groups. The site of action of PAO was localized by photolabeling in the beta-subunit of flavocytochrome b [Doussière, J., Poinas, A, Blais, C., and Vignais, P. V. (1998) Eur. J. Biochem. 251, 649-658]. Moreover, the spin state of heme b is controlled by interaction of arachidonic acid with the flavocytochrome b [Doussière, J., Gaillard, J., and Vignais, P. V. (1996) Biochemistry 35, 13400-13410]. Here we report that the promoting effect of arachidonic acid on the activation of NADPH oxidase is due to specific binding of arachidonic acid to flavocytochrome b. Elicitation of NADPH oxidase activity by arachidonic acid is in part associated with an increased affinity of flavocytochrome b for O(2), an effect that was counteracted by the methyl ester of arachidonic acid. On the other hand, the affinity for NADPH was not affected by arachidonic acid. We further demonstrate that PAO antagonizes the effect of arachidonic acid on oxidase activation by decreasing the affinity of the oxidase for O(2), but not for NADPH. PAO induced a change in the spin state of heme b, as arachidonic acid does, with, however, some differences in the constraints imposed to the heme. It is concluded that the opposite effects of arachidonic acid and PAO are exerted on the beta-subunit of flavocytochrome b at two different interacting sites.

Adenosine Triphosphate↗

PubMed2002

The effect of pH on the alternative oxidase activity in isolated Acanthamoeba castellanii mitochondria.

Mitochondria of Acanthamoeba castellanii possess a cyanide-resistant GMP-stimulated ubiquinol alternative oxidase in addition to the cytochrome pathway. In a previous work it has been observed that an interaction between the two ubiquinol-oxidizing pathways exists in intact A. castellanii mitochondria and that this interaction may be due to a high sensitivity of the alternative oxidase to matrix pH. In this study we have shown that the alternative oxidase activity reveals a pH-dependence with a pH optimum at 6.8 whatever the reducing substrate may be. The GMP stimulation of alternative oxidase is also strongly dependent on pH implicating probably protonation/deprotonation processes at the level of ligand and protein with an optimum pH at 6.8. The ubiquinone redox state-dependence of alternative oxidase activity is modified by pH in such a way that the highest activity for a given ubiquinone redox state is observed at pH 6.8. Thus pH, binding of GMP, and redox state of ubiquinone collaborate to set the activity of the GMP-stimulated alternative oxidase in isolated A. castellanii mitochondria. The high pH sensitivity of the alternative oxidase could link inactivation of the cytochrome pathway proton pumps to activation of the alternative oxidase with acceleration of redox free energy dissipation as a consequence.

Acanthamoeba↗

PubMed1997

Human cytochrome c oxidase: structure, function, and deficiency.

As the terminal component of the mitochondrial respiratory chain, cytochrome c oxidase plays a vital role in cellular energy transformation. Human cytochrome c oxidase is composed of 13 subunits. The three major subunits form the catalytic core and are encoded by mitochondrial DNA (mtDNA). The remaining subunits are nuclear-encoded. The primary sequence is known for all human subunits and the crystal structure of bovine heart cytochrome c oxidase has recently been reported. However, despite this wealth of structural information, the role of the nuclear encoded subunits is still poorly understood. Yeast cytochrome c oxidase is a close model of its human counterpart and provides a means of studying the effects of mutations on the assembly, structure, stability and function of the enzyme complex. Defects in cytochrome c oxidase function are found in a clinically heterogeneous group of disorders. The molecular defects that underlie these diseases may arise from mutations of either mitochondrial or the nuclear genomes or both. A significant number of cytochrome c oxidase deficiencies, often associated with other respiratory chain enzyme defects, are attributed to mutations of mtDNA. Mutations of mtDNA appear, nonetheless, uncommon in early childhood. Pedigree analysis and cell fusion experiments have demonstrated a nuclear involvement in some infantile cases but a specific genomic lesion has not yet been reported. Detailed analyses of the many steps involved in the biogenesis of cytochrome c oxidase, often pioneered in yeast, offer several starting points for further molecular characterizations of cytochrome c oxidase deficiencies observed in clinical practice.

Amino Acid Sequence↗

PubMed2001

Inhibition of the neutrophil NADPH oxidase and associated H+ channel by diethyl pyrocarbonate (DEPC), a histidine-modifying agent: evidence for at least two target sites.

Diethyl pyrocarbonate (DEPC), a histidine-modifying reagent, has been utilized to demonstrate the importance of histidine residues in the functioning of proteins. In previous studies of the NADPH oxidase, histidine residues have been determined to be important in the ability of gp91(phox) to function as an H(+) pathway and in the binding of haem and FAD. We have investigated the ability of DEPC to inhibit H(+) flux and superoxide generation by human neutrophils. Proton flux through the NADPH oxidase-associated H(+) channel was inhibited by DEPC only if applied simultaneously with an activator of the channel. This suggested that the site modified by DEPC is not accessible in the closed channel. Superoxide generation by the NADPH oxidase was also inhibited by DEPC when applied after or simultaneously with the activator. Translocation of the NADPH oxidase cytosolic components, p67(phox) and p47(phox), to the membrane was unaffected by DEPC. In a cell-free system, DEPC-treated membranes failed to support superoxide generation or the reduction of Iodonitrotetrazolium Violet and showed a loss of the characteristic cytochrome b(558) spectrum. Superoxide generation by DEPC-treated cytosol was inhibited slightly. Therefore it can be concluded that there are two sites within the NADPH oxidase that interact with DEPC, one in the H(+) pathway, only accessible in the activated oxidase, and a second accessible prior to activation of the NADPH oxidase. The latter non-proton pathway DEPC site is located within the membrane components of the NADPH oxidase and is associated with the binding of haem in the enzyme complex.

Cytochrome b Group↗

PubMed2002

Involvement of protein kinase D in Fc gamma-receptor activation of the NADPH oxidase in neutrophils.

Protein kinases involved in the activation of the NADPH oxidase by Fc gamma receptors in neutrophils were studied. Of three different protein kinase C (PKC) inhibitors, Gö 6976 inhibited the NADPH oxidase completely, whereas bisindolylmaleimide I and Ro 31-8220 caused a 70-80% inhibition. Thus a Gö 6976-sensitive, bisindolylmaleimide I/Ro 31-8220-insensitive component contributes to NADPH oxidase activation induced by Fc gamma receptors. Down-regulation of PKC isotypes resulted in inhibition of Fc gamma-receptor-activated NADPH oxidase, but a down-regulation-insensitive component was still present. This component was sensitive to Gö 6976, but insensitive to Ro 31-8220. It has been shown previously that protein kinase D/PKC-mu (PKD) shows this same pharmacology in vitro. We show that PKD is present in neutrophils and that, in contrast with PKC isotypes, PKD is not down-regulated. Therefore PKD may participate in NADPH oxidase activation. To obtain direct evidence for this we adopted an antisense approach. Antisense PKD inhibited NADPH oxidase induced by Fc gamma-receptor stimulation by 50% and the Ro 31-8220-insensitive component in the activation was inhibited by antisense PKD. In vitro kinase assays showed that PKD is activated by presenting IgG-opsonized particles to neutrophils. Furthermore, PKD localizes to the area of particle intake in the cell and phosphorylates two of the three cytosolic components of the NADPH oxidase, p40(phox) and p47(phox). Taken together, these data indicate that Fc gamma receptors engage PKD in the regulation of the NADPH oxidase.

Carbazoles↗

PubMed1985

Partial characterization of lysyl oxidase from several human tissues.

Lysyl oxidase activity was assayed in urea extracts of a number of human tissues, proving to be highest in skin. Antibodies to human placental lysyl oxidase completely inhibited the activity of crude lysyl oxidase from all the human tissues studied, with no significant differences in the amounts of antiserum required for 50% inhibition. By contrast, marked differences were found in this value between skin tissue samples from different species. The Mr of lysyl oxidase in crude extracts of human skin and in the medium of cultured human skin fibroblasts was 30 000 by gel filtration, no active species with a higher Mr being detectable. Four forms of lysyl oxidase activity were seen in DEAE-cellulose chromatography of urea extract from human skin, all having Mr 30 000. Antibodies to human placental lysyl oxidase stained a 30 000-Mr protein in urea extracts of all the human tissues studied and in the medium of cultured human skin fibroblasts when examined by immunoblotting after sodium dodecyl sulphate/polyacrylamide-slab-gel electrophoresis, but they also stained high-Mr material. The findings suggest that there are no immunologically distinct lysyl oxidase isoenzymes in the various human tissues and that the true Mr of lysyl oxidase in crude urea extracts is 30 000.

Amino Acid Oxidoreductases↗

PubMed1992

Purification and characterization of polyamine oxidase from Ascaris suum.

The interconversion of polyamines in the parasite nematode Ascaris suum by a novel type of polyamine oxidase was demonstrated. The nematode enzyme was clearly distinguishable from monoamine and diamine oxidases as well as from the mammalian polyamine oxidase, as shown by the use of the specific inhibitors pargyline, aminoguanidine and MDL 72527 respectively. All three inhibitors had no effect on the parasite polyamine oxidase, and the enzyme did not accept diamines such as putrescine, cadaverine or histamine as substrates. The parasite polyamine oxidase selectively oxidizes spermine and spermidine but not N-acetylated polyamines, whereas the mammalian tissue-type polyamine oxidase shows preference for the N-acetylated polyamines. These results suggest a regulatory function of the nematode polyamine oxidase in the degradation and interconversion of polyamines in parasite nematodes. The enzyme was purified to homogeneity by gel filtration, preparative isoelectric focusing and subsequent affinity chromatography on spermine- and berenil-Sepharose 4B. With respect to reaction type, the prosthetic group FAD, the molecular mass (66 kDa) and the contents of thiol and carbonyl groups, the polyamine oxidase from A. suum is similar to the isofunctional enzyme of mammalian tissue.

Animals↗

PubMed1993

Relationship between phosphorylation and translocation to the plasma membrane of p47phox and p67phox and activation of the NADPH oxidase in normal and Ca(2+)-depleted human neutrophils.

Stimulation of neutrophils with different agonists activates a latent multicomponent NADPH oxidase that reduces molecular oxygen to superoxide anion. Evidence has accumulated that phosphorylation of p47phox (the 47 kDa cytosolic phagocyte oxidase factor) and translocation of the two cytosolic components p47phox and p67phox are essential steps in the activation of NADPH oxidase in response to phorbol esters. We analysed the relationships between activation of the NADPH oxidase and phosphorylation and translocation of p47phox and p67phox in normal and Ca(2+)-depleted neutrophils stimulated by the receptor-mediated agonists formyl-methionyl-leucyl-phenylalanine and concanavalin A. The results produced the following conclusions: (1) Translocation of p47phox and p67phox is an essential mechanism for activation of the NADPH oxidase. (2) A continuous translocation of p47phox and p67phox is necessary to maintain the NADPH oxidase in an activated state. (3) Only a fraction of p47phox and p67phox translocated to the plasma membrane is functional for the activation of the oxidase. (4) Translocation is independent of protein kinase C, and is linked to transmembrane signalling involving Ca2+ transients and production of lipidic second messengers. However, under some conditions, such as in Ca(2+)-depleted neutrophils, translocation can also occur independently of signalling pathways involving production of second messengers from hydrolysis of phospholipids and Ca2+ transients. (5) Phosphorylation of p47phox and p67phox can be quantitatively dissociated from translocation, as staurosporine markedly inhibits phosphorylation but not translocation. (6) The activity of NADPH oxidase is not correlated with the amounts of the phosphorylated proteins present in the plasma membrane.

Alkaloids↗

PubMed1990

Renal purine efflux and xanthine oxidase activity during experimental nephrosis in rats: difference between puromycin aminonucleoside and adriamycin nephrosis.

1. The hypothesis was tested that the renal xanthine oxidase system provides a source of oxygen free radicals in puromycin aminonucleoside and adriamycin experimental nephrosis by generating uric acid from hypoxanthine and xanthine. 2. The concentrations in renal tissue of the putative intermediary products of puromycin aminonucleoside metabolism, hypoxanthine and xanthine, and of their precursors, adenosine and inosine, were lower in rats treated with puromycin aminonucleoside than in normal controls, whereas concentrations of the metabolites were normal after adriamycin intoxication. Their daily urinary excretion was lower in the 24 h after puromycin aminonucleoside administration compared with the baseline values and returned to near normal levels within 5 days. After adriamycin the 24 h urinary excretion of xanthine and uric acid was double the baseline levels (P less than 0.001). 3. When equimolar amounts of hypoxanthine were injected instead of puromycin aminonucleoside, the concentration of all bases increased slightly in renal tissue and their urinary efflux was double the baseline level: allantoin, uric acid, the unmodified nucleotide and xanthine were the most represented compounds in urine. 4. The enzymatic activities relative to xanthine oxidase (EC 1.1.3.22) and xanthine dehydrogenase (EC 1.1.1.204) in renal tissues were unchanged 1 day after puromycin aminonucleoside or hypoxanthine intoxication and only moderately increased in both groups at 13 days (the time of appearance of heavy proteinuria in the puromycin aminonucleoside-treated group). In contrast, xanthine oxidase and xanthine dehydrogenase activities were higher in adriamycin-treated rats at 1 and 15 days after the treatment (P less than 0.001). 5. Feeding rats with normoprotein diets containing tungsten induced a marked and constant decrease of renal xanthine oxidase and xanthine dehydrogenase activities to 20% of the baseline values in both puromycin aminonucleoside- and adriamycin-treated rats. Inhibition of renal xanthine oxidase and xanthine dehydrogenase activities by tungsten was associated with a marked reduction (P less than 0.001) of proteinuria in adriamycin-treated rats and the same occurred with allopurinol, a specific inhibitor of xanthine oxidase activity. In contrast, tungsten treatment did not reduce the proteinuria associated with puromycin aminonucleoside, which reached a maximum 13 days after puromycin aminonucleoside intoxication. Hypoxanthine-treated rats were normoproteinuric after 2 months of observation. 6. These data demonstrate an activation of renal xanthine oxidase and xanthine dehydrogenase after adriamycin intoxication which is relevant to the induction of proteinuria. They also argue against the involvement of the renal xanthine oxidase system as a source of free radicals in puromycin aminonucleoside nephrosis and suggest that the nucleotide cycle is not a normal route for puromycin aminonucleoside degradation.(ABSTRACT TRUNCATED AT 400 WORDS)

Allantoin↗

PubMed1992

Absolute requirement for GTP in activation of human neutrophil NADPH oxidase in a cell-free system: role of ATP in regenerating GTP.

Guanine and/or adenine nucleotides appear to be involved in the activation of the superoxide-generating NADPH oxidase of phagocytic cells. Their precise roles, however, are unclear, as much of the evidence for their involvement comes from experiments in which nucleotides have been added to complex systems already rich in both endogenous nucleotides and enzymes capable of interconverting them. To circumvent this problem we have examined the role of nucleotides in neutrophil NADPH oxidase activation by using a cell-free system in which adenine and guanine nucleotide concentrations were carefully controlled and monitored by (i) depletion of endogenous nucleotides by extensive dialysis and charcoal treatment; (ii) reconstitution of the depleted system with reagents analyzed for purity; and (iii) measurement of nucleotide levels in cytosol preparations and in oxidase reaction mixtures by HPLC analysis. In contrast to previous reports that have demonstrated only a several-fold enhancement of oxidase activity by GTP or its analogs, we have shown that oxidase activation was absolutely dependent upon GTP in reactions containing dialyzed cytosol in which the total endogenous nucleotide levels were reduced by greater than 99.5%. Kinetic studies revealed that GTP is required at or before the rate-limiting step in oxidase activation. Two nonhydrolyzable analogs of GTP, guanosine 5'-(gamma-thio)triphosphate and guanylyl imidodiphosphate, were even more active than GTP, suggesting the involvement of one or more GTP-binding proteins. In contrast, ATP was neither necessary nor sufficient for oxidase activation. If reaction mixtures were contaminated with GDP and/or GMP, however, ATP (but not its nonhydrolyzable analog adenylyl imidodiphosphate) could indirectly support oxidase activation by means of endogenous enzymes that catalyze the ATP-dependent conversion of GMP and GDP to GTP.

Adenine Nucleotides↗

PubMed1995

Translocation of p21rac2 from cytosol to plasma membrane is neither necessary nor sufficient for neutrophil NADPH oxidase activity.

Activation of the membrane-associated NADPH oxidase of neutrophils requires several cytosolic factors including p47phox, p67phox and p21rac2. We compared NADPH oxidase activity with the membrane translocation of p47phox, p67phox, and p21rac2. In a cell-free system, GTP gamma S stimulated translocation of p47phox and p67phox to the plasma membrane only in the presence of arachidonate, and this translocation correlated with NADPH oxidase activity of the reisolated plasma membranes (R = 0.94 and 0.97, respectively). In contrast, GTP gamma S-stimulated p21rac2 translocation with or without arachidonate, and the extent of translocation did not correlate with oxidase activity (R = 0.17). Neutrophil cytoplasts were used to relate membrane translocation of p47phox, p67phox and p21rac2 to membrane oxidase activity in response to the inflammatory agonists. Whereas N-formyl-methionyl-leucyl-phenylalanine stimulated equimolar, transient membrane translocation of p47phox and p67phox which kinetically paralleled NADPH oxidase activity, relatively little p21rac2 translocated (moles of p47phox/p21rac2 = 16.6). Moreover, although phorbol 12-myristate 13-acetate stimulated both the stable translocation of p47phox and p67phox and sustained NADPH oxidase activity, it did not stimulate p21rac2 translocation. From these data we conclude that membrane translocation of p21rac2 does not regulate NADPH oxidase activity stoichiometrically.

Cell Fractionation↗

PubMed1995

Substrate control of free radical generation from xanthine oxidase in the postischemic heart.

While the free radical-generating enzyme xanthine oxidase is a central mechanism of injury in postischemic tissues, questions remain regarding how xanthine oxidase-mediated radical generation is triggered during ischemia and reperfusion. There is controversy regarding whether radical generation is caused by enzyme formation of that of its substrates xanthine and hypoxanthine. Therefore, studies were performed in isolated rat hearts correlating the magnitude and time course of radical generation with alteration in xanthine oxidase and its substrates. Radical generation was measured by electron paramagnetic resonance spectroscopy and correlated with spectrophotometric assays of tissue xanthine oxidase activity and chromatographic measurements of tissue and effluent concentrations of xanthine oxidase substrates and products. Xanthine oxidase was present in preischemic hearts and slightly increased during 30-min global ischemia. Hypoxanthine and xanthine were not present prior to ischemia but accumulated greatly during ischemia due to ATP degradation. These substrate concentrations rapidly declined over the first 5 min of reperfusion matching the observed time course of radical generation, whereas xanthine oxidase activity was largely unchanged. Both substrates were also observed in the coronary effluent during the first 5 min of reflow along with the product uric acid. Thus, the burst of xanthine oxidase-mediated free radical generation upon reperfusion is triggered and its time course controlled by a large increase in substrate formation that occurs secondary to the degradation of ATP during ischemia.

Adenosine Triphosphate↗