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PubMed2001

Involvement of NADH/NADPH oxidase in human platelet ROS production.

Platelets play an important role in atherosclerotic and thromboembolic vascular diseases. It has been reported that reactive oxygen species (ROS) could modify platelet function, and platelets themselves have the ability to produce ROS. However, the enzymatic sources of ROS in platelets have not been fully determined. The NADH/NADPH oxidase system was originally identified as the major source of ROS in phagocytes. Recently, it has become evident that this oxidase is functionally expressed not only in phagocytes but also in various cell types. The present study was undertaken to test the hypothesis that NADH/NADPH oxidase might be expressed in human platelets. Lucigenin-enhanced chemiluminescence (L-CL) and electron spin resonance (ESR) method demonstrated that human platelets obtained from healthy volunteers released ROS, and the released ROS were increased by stimulation with 12-O-tetradecanoylphorbol-13-acetate (TPA) or calcium ionophore. Homogenates of human platelets, as well as MEG01 cells, megakaryocytic cell line, had the enzymatic activity to produce superoxide in NADH/NADPH-dependent manners. This enzymatic activity was suppressed by diphenylene iodonium (DPI), an inhibitor of NADH/NADPH oxidase. Western blot analysis demonstrated that platelets and MEG01 cells expressed p22(phox) and p67(phox) proteins, components of NADH/NADPH oxidase. Thus, human platelets have the enzymatic activity of p22(phox)-based NADH/NADPH oxidase, and this oxidase is likely one of the important sources of ROS in platelets.

Blood Platelets↗
PubMed2000

Reaction of nitric oxide with the turnover intermediates of cytochrome c oxidase: reaction pathway and functional effects.

The reactions of nitric oxide (NO) with the turnover intermediates of cytochrome c oxidase were investigated by combining amperometric and spectroscopic techniques. We show that the complex of nitrite with the oxidized enzyme (O) is obtained by reaction of both the "peroxy" (P) and "ferryl" (F) intermediates with stoichiometric NO, following a common reaction pathway consistent with P being an oxo-ferryl adduct. Similarly to chloride-free O, NO reacted with P and F more slowly [k approximately (2-8) x 10(4) M(-1) s(-1)] than with the reduced enzyme (k approximately 1 x 10(8) M(-1) s(-1)). Recovery of activity of the nitrite-inhibited oxidase, either during turnover or after a reduction-oxygenation cycle, was much more rapid than nitrite dissociation from the fully oxidized enzyme (t(1/2) approximately 80 min). The anaerobic reduction of nitrite-inhibited oxidase produced the fully reduced but uncomplexed enzyme, suggesting that reversal of inhibition occurs in turnover via nitrite dissociation from the cytochrome a(3)-Cu(B) site: this finding supports the hypothesis that oxidase may have a physiological role in the degradation of NO into nitrite. Kinetic simulations suggest that the probability for NO to be transformed into nitrite is greater at low electron flux through oxidase, while at high flux the fully reduced (photosensitive) NO-bound oxidase is formed; this is fully consistent with our recent finding that light releases the inhibition of oxidase by NO only at higher reductant pressure [Sarti, P., et al. (2000) Biochem. Biophys. Res. Commun. 274, 183].

Animals↗
PubMed1995

Involvement of cytochrome c oxidase subunit III in energy coupling.

The role of the conserved acidic residues of subunit III of cytochrome c oxidase (COIII) in energy transduction was investigated. Using a COIII deletion mutant of Paracoccus denitrificans, complemented with a plasmid expressing either the wild type (wt) COIII gene or site-directed mutants of the COIII gene, we measured cytochrome c oxidase-dependent ATP synthesis, respiration, and membrane potential. Cytochrome c oxidase-dependent ATP synthesis was attenuated in nonacidic mutants of either Glu98 (E98A and E98Q), or Asp259 (D259A) but not in the acidic mutant E98D. The rates of respiration in the energy conversion-defective mutants were as high as or higher than that in the wt. The cytochrome c oxidase-induced increment of membrane potential in the nonacidic mutants was similar to or higher than that in the wt. In contrast, when succinate-driven ATP synthesis was mediated solely by ubiquinol oxidase (e.g., in the presence of myxothiazol), the rates of ATP synthesis in the nonacidic mutants were higher than that in the wt. Moreover, myxothiazol, which inhibited succinate respiration as well as ATP synthesis in wt and E98D, stimulated ATP synthesis, while inhibiting succinate respiration, in the nonacidic mutants. These results indicate that the attenuation of energy conversion in these mutants is limited to cytochrome c oxidase and thus suggest that subunit III plays a role in energy conversion by cytochrome c oxidase.

Adenosine Triphosphate↗
PubMed1993

Comparative resonance Raman study of cytochrome c oxidase from beef heart and Paracoccus denitrificans.

Well-resolved, Soret band excited resonance Raman spectra were measured from the fully oxidized and fully reduced cytochrome c oxidase from beef heart and Paracoccus denitrificans. The vibrational patterns in the marker band region (1450-1700 cm-1) were analyzed, and a complete assignment of heme a and heme a3 vibrational modes is presented, permitting a detailed structural comparison of the mammalian and bacterial enzymes. Similar frequencies of the porphyrin modes for the reduced heme a and the reduced and oxidized heme a3 are found, indicating a close relationship of the ground-state conformations in all oxidase species studied. In oxidized heme a, however, significant frequency differences are observed and interpreted in terms of a ruffled porphyrin structure in the three- and two-subunit forms of the Paracoccus enzyme compared to the planar heme a of beef heart oxidase. The structural distortions, which also perturb the conformation of the formyl substituent and its electronic coupling with the porphyrin, reflect the specific heme-protein interactions at heme a. Since in the fully reduced state heme a appears to be largely planar in all oxidase species, the redox-linked conformational transition requires a more drastic rearrangement of the heme a-protein interactions in the bacterial than in the mammalian oxidase. For both heme a and heme a3 in the reduced state and for heme a3 in the oxidize state, frequency, intensity, and bandwidth differences of the formyl stretching vibration and intensity differences of some porphyrin modes are noted between the three oxidase forms. The same modes are also affected by quaternary structure changes in the bovine oxidase caused by different detergents and isolation procedures. These effects are attributed to differences of the dielectric properties of the heme environment, due to subtle structural changes in the heme pockets, induced by protein-protein interactions of subunit III with subunits I and/or II.

Animals↗
PubMed1985

Protein and lipid structural transitions in cytochrome c oxidase-dimyristoylphosphatidylcholine reconstitutions.

The thermotropic behavior of the mitochondrial enzyme cytochrome c oxidase (EC 1.9.3.1) reconstituted in dimyristoylphosphatidylcholine (DMPC) vesicles has been studied by using high-sensitivity differential scanning calorimetry and fluorescence spectroscopy. The incorporation of cytochrome c oxidase into the phospholipid bilayer perturbs the thermodynamic parameters associated with the lipid phase transition in a manner analogous to other integral membrane proteins: it reduces the enthalpy change, lowers the transition temperature, and reduces the cooperative behavior of the phospholipid molecules. Analysis of the dependence of the enthalpy change on the protein:lipid molar ratio indicates that cytochrome c oxidase prevents 99 +/- 5 lipid molecules from participating in the main gel-liquid-crystalline transition. These phospholipid molecules presumably remain in the same physical state below and above the transition temperature of the bulk lipid, thus providing a more or less constant microenvironment to the protein molecule. The effect of the phospholipid bilayer matrix on the thermodynamic stability of the cytochrome c oxidase complex was examined by high-sensitivity differential scanning calorimetry. Detergent (Tween 80)-solubilized cytochrome c oxidase undergoes a complex, irreversible thermal denaturation process centered at 56 degrees C and characterized by an enthalpy change of 550 +/- 50 kcal/mol of enzyme complex. Reconstitution of the cytochrome c oxidase complex into DMPC vesicles shifts the transition temperature upward to 63 degrees C, indicating that the phospholipid bilayer moiety stabilizes the native conformation of the enzyme. The lipid bilayer environment contributes approximately 10 kcal/mol to the free energy of stabilization of the enzyme complex. The thermal unfolding of cytochrome c oxidase is not a two-state process.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Cytosolic factors in bovine neutrophil oxidase activation. Partial purification and demonstration of translocation to a membrane fraction.

The O2(.-)-generating oxidase of bovine neutrophils is activated in a cell-free system consisting of a particulate fraction enriched in plasma membrane and containing the dormant oxidase, a high-speed supernatant from neutrophil homogenate (cytosol), Mg ions, GTP gamma S, and arachidonic acid [Ligeti, E., Doussiere, J., & Vignais, P.V. (1988) Biochemistry 27, 193-200]. The cytosolic components participating in the activation of the membrane-bound oxidase have been investigated. These components were resolved into several active peaks by Q Sepharose chromatography. The oxidase-activating potency of these peaks was synergistically enhanced by combining samples from separate peaks, or by supplying them with a threshold amount of crude cytosol. Partial purification of two active fractions containing a limited number of proteins of 65, 56, 53, and 45 kDa was achieved by gel filtration of cytosol on Ultrogel AcA44, followed by chromatography on hydroxylapatite and Mono Q. The specific oxidase-activating potency of these partially purified fractions (activating potency per milligram of soluble protein) was 6-8-fold higher than that of crude cytosol; it was enhanced up to 75-fold by complementation with a minute amount of crude cytosol, which per se had a limited efficiency. These data indicate that oxidase activation requires more than one cytosolic component to be activated. To check whether translocation of cytosolic proteins to the membrane occurred concomitantly with oxidase activation, use was made of radiolabeled cytosolic proteins. Cytosol was treated with phenyl[14C]isothiocyanate ([14C]PITC), such that 60% of its activation potency was still present.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2004

Portability of oxidase domains in nonribosomal peptide synthetase modules.

Oxazole and thiazole rings are present in numerous nonribosomal peptide natural products. Oxidase domains are responsible for catalyzing the oxidation of thiazolines and oxazolines to yield fully aromatic heterocycles. Unlike most domains, the placement of oxidase domains within assembly line modules varies. Noting this tolerance, we investigated the portability of an oxidase domain to a heterologous assembly line. The epimerase domain of PchE, involved in pyochelin biosynthesis, was replaced with the oxidase domain from MtaD, involved in myxothiazol biosynthesis. The chimeric module was expressed in soluble form as a flavin mononucleotide-containing flavoprotein. The functionality of the inserted oxidase domain was assayed within PchE and in transfer of the growing siderophore acyl chain from PchE to the next downstream module. While pyochelin-like product release was not observed downstream, the robust activity of the transplanted oxidase domain and the ability of the chimeric module to produce an advanced intermediate bound to the synthetase underscore the possibility of future engineering within nonribosomal peptide synthetase pathways using oxidase domains.

Bacterial Proteins↗
PubMed2006

pH dependence of proton translocation in the oxidative and reductive phases of the catalytic cycle of cytochrome c oxidase. The role of H2O produced at the oxygen-reduction site.

A study is presented on the pH dependence of proton translocation in the oxidative and reductive phases of the catalytic cycle of purified cytochrome c oxidase (COX) from beef heart reconstituted in phospholipid vesicles (COV). Protons were shown to be released from COV both in the oxidative and reductive phases. In the oxidation by O2 of the fully reduced oxidase, the H+/COX ratio for proton release from COV (R --> O transition) decreased from approximately 2.4 at pH 6.5 to approximately 1.8 at pH 8.5. In the direct reduction of the fully oxidized enzyme (O --> R transition), the H+/COX ratio for proton release from COV increased from approximately 0.3 at pH 6.5 to approximately 1.6 at pH 8.5. Anaerobic oxidation by ferricyanide of the fully reduced oxidase, reconstituted in COV or in the soluble case, resulted in H+ release which exhibited, in both cases, an H+/COX ratio of 1.7-1.9 in the pH range 6.5-8.5. This H+ release associated with ferricyanide oxidation of the oxidase, in the absence of oxygen, originates evidently from deprotonation of acidic groups in the enzyme cooperatively linked to the redox state of the metal centers (redox Bohr protons). The additional H+ release (O2 versus ferricyanide oxidation) approaching 1 H+/COX at pH < or = 6.5 is associated with the reduction of O2 by the reduced metal centers. At pH > or = 8.5, this additional proton release takes place in the reductive phase of the catalytic cycle of the oxidase. The H+/COX ratio for proton release from COV in the overall catalytic cycle, oxidation by O2 of the fully reduced oxidase directly followed by re-reduction (R --> O --> R transition), exhibited a bell-shaped pH dependence approaching 4 at pH 7.2. A mechanism for the involvement in the proton pump of the oxidase of H+/e- cooperative coupling at the metal centers (redox Bohr effects) and protonmotive steps of reduction of O2 to H2O is presented.

Anaerobiosis↗
PubMed2006

Sequence analysis of the cbb3 oxidases and an atomic model for the Rhodobacter sphaeroides enzyme.

The cbb3-type oxidases are members of the heme-copper oxidase superfamily, distant by sequence comparisons, but sharing common functional characteristics. To understand the minimal common properties of the superfamily, and to learn about cbb3-type oxidases specifically, we have analyzed a wide set of heme-copper oxidase sequences and built a homology model of the catalytic subunit of the cbb3 oxidase from Rhodobacter sphaeroides. We conclude that with regard to the active site surroundings, the cbb3 oxidases greatly resemble the structurally known oxidases, while major differences are found in three segments: the additional N-terminal stretch of ca. 60 amino acids, the segment following helix 3 to the end of helix 5, and the C-terminus from helix 11 onward. The conserved core contains the active site tyrosine and also an analogue of the K-channel of proton transfer, but centered on a well-conserved histidine in the lower part of helix 7. Modeling the variant parts of the enzyme suggests that two periplasmic loops (between helices 3 and 4 and between helices 11 and 12) could interact with each other as a part of the active site structure and might have an important role in proton pumping. An analogue of the D-channel is not found, but an alternative channel might form around helix 9. A preliminary packing model of the trimeric enzyme is also presented.

Amino Acid Sequence↗
PubMed1999

The cbb3 terminal oxidase of Rhodobacter sphaeroides 2.4.1: structural and functional implications for the regulation of spectral complex formation.

We have previously shown that the flow of reductant through the cbb3 terminal cytochrome c oxidase of Rhodobacter sphaeroides is essential to the repression of photosynthesis (PS) gene expression in the presence of oxygen by inhibiting the functional activity of the Prr two-component activation system. To gain further insight into the role of the cbb3 oxidase and the cognate ccoNOQP operon in the oxygen regulation of PS gene expression, we constructed nonpolar, in-frame deletions within the ccoN and ccoQ genes. Whereas mutations in ccoN, ccoQ, and ccoP resulted in PS gene expression in the presence of oxygen, only the ccoQ mutation showed both the normal flow of reductant through the cbb3 oxidase and the absence of any alteration in the relative levels of spheroidene and spheroidenone, as is observed for those mutations in the cco operon that result in the loss of terminal oxidase activity. Consistent with these findings is the observation that extra copies of the ccoNOQP operon in trans resulted in the decreased formation of both the B800-850 and B875 spectral complexes under anaerobic growth conditions. These results in conjunction with our earlier findings indicate that (1) the flow of reductant through the cbb3 terminal oxidase is a prerequisite to the regulation of PS gene expression by the Prr two-component regulatory system, (2) the CcoQ protein is involved in conveying the signal derived from reductant flow through the cbb3 terminal oxidase to the Prr regulatory pathway, (3) there is reductant flow through this terminal oxidase under anaerobic conditions, and as a result, the activity of the Prr system is still subject to cbb3 regulation, and (4) the acceptor for reductant flow through cbb3 under anaerobic conditions is in whole or in part involved in the conversion of spheroidene to spheroidenone.

Carotenoids↗
PubMed2001

Cytochrome oxidase as an indicator of ice storage and frozen storage.

The potential of cytochrome oxidase as an indicator of ice storage and frozen storage of fish was investigated. Optimal assay conditions for cytochrome oxidase in a crude homogenate from cod muscle were studied. Maximal cytochrome oxidase activity was found at pH 6.5-7.5 and an assay temperature of 30 degrees C. Maximal activation by Triton X-100 was obtained in a range of 0.62-1.25 mM Triton X-100. The specificity of the assay was high, as cytochrome oxidase was inhibited 98% by 33 microM of the specific inhibitor sodium azide. The coefficient of variation of cytochrome oxidase activity in different cods was 21%, and the coefficient of variation of different analyses on the same homogenate was 5%. It was shown that ice storage of muscle samples before they were frozen and thawed resulted in a major freezing-induced activation of cytochrome oxidase activity. The enzyme may therefore be used as an indicator of frozen fish to determine if the fish has been stored on ice before freezing. Cytochrome oxidase activity showed also potential as an indicator of frozen storage, as it was possible to distinguish between the frozen storage temperatures -9, -20, and -40 degrees C.

Animals↗
PubMed2004

Identification of a genomic clone to ACC oxidase from papaya (Carica papaya L.) and expression studies.

In this paper are presented structural analysis and expression studies of one genomic clone encoding a 1-aminocyclopropane-1-carboxylate oxidase (ACC oxidase) from papaya. Using RT-PCR amplification of ACC oxidase cDNAs from ripe papaya, a product of 800 bp was obtained, which after sequence analysis was found to code for a protein highly homologous to ACC oxidase proteins. This PCR product was used as a probe for screening a genomic library, and two different groups of clones were obtained as indicated by restriction mapping. One clone (CPACCO-1) was selected for further study and fully sequenced. Comparison of this sequence with the PCR product and other cloned ACC oxidase genes revealed that CPACCO-1 encoded the transcript in four exons interrupted by three introns. Southern blot analysis showed one or two major bands hybridized to the PCR probe, suggesting that the ACC oxidase gene is present in one or two copies in the papaya genome. By northern blot analysis it was found that the ACC oxidase transcripts appear in the pulp earlier than in the peel, suggesting a developmental regulation. A wounding experiment revealed the highest expression of this gene by 2 h. Transcriptional regulation by ethylene could be due to the presence of a putative GCC box in the promoter region.

Amino Acid Oxidoreductases↗
PubMed2000

Roles of respiratory oxidases in protecting Escherichia coli K12 from oxidative stress.

Isogenic strains of Escherichia coli that were defective in either of the two major aerobic terminal respiratory oxidases (cytochromes bo' and bd) or in the putative third oxidase (cytochrome bd-II) were studied to elucidate role(s) for oxidases in protecting cells from oxidative stress in the form of H2O2 and paraquat. Exponential phase cultures of all three oxidase mutants exhibited a greater decline in cell viability when exposed to H2O2 stress compared to the isogenic parent wild-type strain. Cytochrome bo' mutants showed the greatest sensitivity to H2O2 under all conditions studied indicating that this oxidase was crucial for protection from H2O2 in E. coli. Cell killing of all oxidase mutants by H2O2 was by an uncharacterized mechanism (mode 2 killing) with cell growth rate affected. The expression of phi(katG-lacZ), an indicator of intracellular H2O2, was 2-fold higher in a cydAB::kan mutant compared to the wild-type strain at low H2O2 concentrations (< 100 microM) suggesting that cytochrome bd mutants were experiencing higher intracellular levels of H2O2. Protein fusions to the three oxidase genes demonstrated that expression of genes encoding cytochrome bd, but not cytochrome bo' or cytochrome bd-II was increased in the presence of external H2O2. This increase in expression of 4P(cydA-lacZ) by H2O2 was further enhanced in a cyo::kan mutant. The level of cytochrome bd determined spectrally and phi(cydA-lacZ) expression was 5-fold and 2-fold higher respectively in an rpoS mutant compared to isogenic wild-type cells suggesting that RpoS was a negative regulator of cytochrome bd. Whether the effect of RpoS is direct or indirect remains to be determined.

Bacterial Proteins↗
PubMed2000

Heme centers of Rhodothermus marinus respiratory chain. Characterization of its cbb3 oxidase.

Rhodothermus (R.) marinus, a thermohalophilic gram-negative, and strict aerobic bacterium, has a rather distinct respiratory chain, containing a caa3 terminal oxidase, a novel cytochrome bc complex and a HiPIP, which is an electron carrier between this complex and a terminal oxidase (Pereira et al (1999a, c). To further elucidate this unusual respiratory system, its membrane-bound heme centers were characterized by visible and EPR spectroscopies as well as by redox potentiometry. Rhodothermus marinus contains mostly B- and C-type hemes; a small amount of A-type heme is also detected. The heme centers have relatively low reduction potentials, ranging from ca. +250 to -60 mV, at pH 7. A Rieske-type center was not detected, suggesting the absence of a canonical complex III. The major terminal oxidase expressed by R. marinus is a cbb3-type oxidase. Its presence is in agreement with molecular biology studies, which reveal the existence of a gene encoding for a FixN-type oxidase. The oxidase was partially purified and appears to have five subunits, with apparent molecular masses of 64, 57, 36, 26 (C-type heme subunit), and 13 kDa. It contains two low-spin heme C centers, one high-, and one low-spin heme B centers. A full description of the equilibrium redox behavior of the heme centers was obtained for a cbb3 oxidase for the first time. The optical spectrum for each heme center and the corresponding reduction potentials were determined at pH 7: + 195 (heme C), +120 (heme B), -50 (heme C), and -50 mV (heme B3).

Amino Acid Sequence↗
PubMed1997

Inhibition of plasma membrane NADH oxidase activity and growth of HeLa cells by natural and synthetic retinoids.

Several retinoids, both natural and synthetic, were evaluated for their ability to modulate NADH oxidase activity of plasma membranes of cultured HeLa cells and the growth of HeLa cells in culture. Both NADH oxidase activity and the growth of cells were inhibited by the naturally-occurring retinoids all trans-retinoic acid (tretinoin) and retinol as well as by the synthetic retinoids, trans-acitretin, 13-cis-acitretin, etretinate and arotonoid ethylester (Ro 13-6298). For all retinoids tested, inhibition of NADH oxidase activity and inhibition of growth were correlated closely. With tretinoin, etretinate and arotonoid ethylester, NADH oxidase activity and cell growth were inhibited in parallel in proportion to the logarithm of retinoid concentration over the range of concentrations 10(-8) to 10(-5) M. Approximately 70% inhibition of both NADH oxidase activity and growth was reached at 10 microM. With retinol, trans-acitretin and 13-cis-acitretin, inhibition of NADH oxidase activity and growth also were correlated but maximum inhibition of both was about 40% at 10 microM. The possibility is suggested that inhibition of the plasma membrane NADH oxidase activity by retinoids may be related to their mechanism of inhibition of growth of HeLa cells in culture.

Animals↗
PubMed1999

Elevated oxidase and esterase levels associated with permethrin tolerance in Anopheles gambiae from Kenyan villages using permethrin-impregnated nets.

The permethrin tolerance (PT) of a population of the mosquito Anopheles gambiae (Diptera: Culicidae) increased following the introduction of permethrin-impregnated nets for malaria control in certain villages near Kisumu, western Kenya. Using a biochemical test that indirectly measures oxidases associated with permethrin resistance, we found that this population had higher oxidase levels than a comparison population from villages without impregnated nets. Mosquitoes from a colony of An. gambiae selected for PT, the RSP (reduced susceptibility to permethrin) strain, were exposed to permethrin with or without the oxidase inhibitor piperonyl butoxide (PB). Significantly higher mortality rates occurred when permethrin was synergized by PB, presumably by suppression of oxidases responsible for PT. An unselected (UNS) colony of An. gambiae that was more susceptible than RSP in a permethrin-susceptibility bioassay (i.e. LT50 22 min for UNS, vs. 42min for RSP) was compared with the RSP colony for levels of oxidases and esterases. The levels of both enzymes were very significantly higher in the RSP strain (P<0.0001). We speculate that use of impregnated nets selected for higher oxidase and esterase levels in An. gambiae to metabolize permethrin acquired from the nets. Both oxidase and esterase mechanisms could confer cross-resistance to other pyrethroids.

Animals↗
PubMed1996

Differential expression of the 1-aminocyclopropane-1-carboxylate oxidase gene family of tomato.

The tomato ACC oxidase gene family is comprised of three members designated AC01, AC02 and AC03. These are highly homologous throughout the protein coding regions but do show a degree of sequence divergence within the 3' untranslated regions. These regions have been cloned and used as gene-specific probes to analyse the differential expression of the tomato ACC oxidase gene family in various tissues at different stages of development. Results indicate that all three genes are transcriptionally active and display a high degree of inducibility in a number of organs at various stages of the life cycle. Both AC01 and Ac03 transcripts accumulate during the senescence of leaves, fruit and flowers. In addition, it appears that AC01 is wound-inducible in leaves. All three ACC oxidase genes are expressed during flower development, with each showing a temporally distinct pattern of accumulation. In addition, the ACC oxidase transcripts are also spatially regulated throughout flower development; AC01 is predominantly expressed in the petals and the stigma and style, AC02 expression is mainly restricted to tissues associated with the anther cone whereas AC03 transcripts accumulate in all of the floral organs examined apart from the sepals. ACC oxidase enzyme assays and Western blot analysis indicate that both enzyme activity and ACC oxidase protein increase with transcript abundance in several tissues. The physiological role of the differential expression of the ACC oxidase gene family, in relation to the regulation of ethylene synthesis, during these various developmental processes is discussed.

Aging↗
PubMed2002

Inhibition of oxidative stress produced by plasma membrane NADH oxidase delays low-potassium-induced apoptosis of cerebellar granule cells.

From 1 to 3 h after the onset of cerebellar granule cells (CGC) apoptosis in a low-K+(5 mm KCl) medium there was a large decay of NADH and a 2.5-fold increase of the rate of reactive oxygen species (ROS) production (measured using CGC loaded with dichlorodihydrofluorescein). During the same time period, the ascorbate-dependent NADH oxidase activity, which accounted for more than 90% of both total NADH oxidase activity and NADH-dependent *O2- production of CGC lysates, increased 2.5- to threefold. The stimulation of the ascorbate-dependent NADH oxidase activity by oxidized cytochrome c, 2.5-fold at saturation with a K(0.5) of 4-5 microm cytochrome c, can at least partially explain this activation. The plasma membrane ascorbate-dependent NADH oxidase activity accounted for more than 70% of the total activity (both in terms of NADH oxidase and *O2- release) of CGC lysates. 4-Hydroxyquinazoline (4-HQ), which was found to block this apoptotic process, prevented the increase of ROS production. 4-HQ protection against cell viability loss and DNA fragmentation correlated with the inhibition by 4-HQ of the ascorbate-dependent NADH oxidase activity of CGC lysates, showing the same K(0.5)-value (4-5 mm 4-HQ). The efficient blockade of CGC apoptosis by addition of superoxide dismutase to the medium further supports the neurotoxic role of *O2- overproduction by the plasma membrane ascorbate-dependent NADH oxidase.

Animals↗