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PubMed1991

Identification and purification of a peroxisomal branched chain fatty acyl-CoA oxidase.

Isoprenoid (branched) fatty acids such as pristanic acid can be degraded via beta-oxidation in peroxisomes. We synthesized 2-methylpalmitoyl-CoA as a model substrate in order to study the first step of the peroxisomal beta-oxidation of branched fatty acids, catalyzed by an acyl-CoA oxidase. 2-Methylpalmitoyl-CoA oxidase activity was found in rat liver homogenates. Subcellular fractionation demonstrated that the oxidase was confined to peroxisomes. 2-Methylpalmitoyl-CoA oxidase was also present in kidney and intestine. It was not induced in liver or in the extrahepatic tissues by treatment of rats with peroxisome proliferators or by feeding diets containing excess isoprenoids. The enzyme was partially purified together with palmitoyl-CoA oxidase and trihydroxycoprostanoyl-CoA oxidase by heat treatment and ammonium sulfate fractionation of liver extracts. The partially purified preparation was chromatographed on various columns. 2-Methylpalmitoyl-CoA oxidase could be separated from the inducible (by peroxisome proliferators) palmitoyl-CoA oxidase and from trihydroxycoprostanoyl-CoA oxidase, but it always coeluted with the noninducible palmitoyl-CoA oxidase, recently described by us (Schepers, L., Van Veldhoven, P. P., Casteels, M., Eyssen, H. J., and Mannaerts, G. P. (1990) J. Biol. Chem. 265, 5242-5246). 2-Methylpalmitoyl-CoA oxidase was purified to near homogeneity in three chromatographic steps (anion exchange, hydroxylapatite, and gel filtration). Its apparent molecular mass is approximately 415 kDa, and it consists of identical subunits of approximately 70 kDa. The enzyme oxidized 2-methylpalmitoyl-CoA twice as rapidly as palmitoyl-CoA and pristanoyl-CoA as rapidly as palmitoyl-CoA, so that it can be considered as a branched fatty acyl-CoA oxidase. Since pristanoyl-CoA is one of its naturally occurring substrates we propose to name this enzyme pristanoyl-CoA oxidase.

Animals↗
PubMed1989

Effects of sonication and growth temperature on the cytochrome oxidase activity of Bacillus species.

The cytochrome oxidase activity of Bacillus strains was analyzed quantitatively by colorimetric N, N, N', N'-tetramethyl-p-phenylene-diamine (TMPD) oxidase assay. Twenty six type strains were readily grouped, the oxidase positive, the oxidase negative, and the oxidase indeterminate groups. TMPD-dependent oxidase activities for whole cells and sonicated cells were compared. Spectral absorbance analyses of membrane fractions showed that all 26 Bacillus type strains contained the o-type cytochrome oxidase; 19 strains contained cytochrome a + a3 and among which 5 strains contained cytochromed. All of the 10 thermophilic Bacillus strains, which were predominantly oxidase positive, exhibited maximum oxidase activity when grown at optimum temperature (65 degrees C). The other three "caldo-active" thermophiles exhibited maximum oxidase activities when grown at their optimum temperatures, 70 degrees C to 80 degrees C. Mesophilic B. cereus and B. subtilis exhibited maximum oxidase activity in cells grown at 42 degrees C and 55 degrees C, respectively. In no case did growth temperature induce oxidase negative strains to exhibit an oxidase positive reaction, and vice versa.

Bacillus↗
PubMed1982

Quantitation of rat liver xanthine oxidase by radioimmunoassay. A mechanism for sex-specific differences.

To further delineate the mechanism responsible for the differences in xanthine oxidase activity in male and female Sprague-Dawley rats, a sensitive and specific radioimmunoassay (RIA) was developed for the measurement of hepatic xanthine oxidase. The RIA could detect as little as 5 mg of liver enzyme. Specificity of the RIA was confirmed by 1) Ouchterlony double immuno-diffusion in which a single precipitin band exhibited xanthine oxidase activity, when crude liver homogenate and an enzyme-specific stain were used; 2) parallelism between purified 125I-labeled xanthine oxidase and serial dilutions of crude liver homogenate; 3) a linear correlation between xanthine oxidase activity and the level of enzyme protein; and 4) a single protein band coincident with purified xanthine oxidase, when an immunoprecipitate prepared from antisera and crude liver homogenate was analyzed on sodium dodecyl sulfate (SDS) polyacrylamide gels. Whether xanthine oxidase activity was assayed in the absence of nicotinamide adenine dinucleotide (NAD+) (oxidase form) or in the presence of NAD+ (dehydrogenase), male values were consistently higher, and both forms of the enzyme correlated significantly with each other. When purified to homogeneity, neither form of the enzyme was appreciably affected by 17 beta-estradiol or testosterone propionate. When the RIA was employed, levels of hepatic xanthine oxidase were significantly greater in male than in female rats. We concluded from these data that increased xanthine oxidase activity in the male corresponds to a greater quantitative complement of xanthine oxidase protein. Furthermore, lower xanthine oxidase activity in the female cannot be explained by immunologically cross-reactive material without enzyme activity nor by a direct sex-steroid enzyme interaction.

Animals↗
PubMed1996

The proportion of xanthine oxidase activity of total xanthine oxidoreductase activity in situ remains constant in rat liver under various (patho)physiological conditions.

Activity of xanthine oxidoreductase (total xanthine dehydrogenase plus xanthine oxidase) and xanthine oxidase was determined cytophotometrically in periportal and pericentral areas of livers of rats under various (patho)physiological conditions that are known to affect the content of reduced glutathione. For this purpose, rats were either normally fed or fasted for 24 hours, fasted for 24 hours, and treated with diethylmaleate that depleted glutathione or treated by in vivo ischemia for 2 hours in the livers. Xanthine oxidoreductase activity was shown histochemically with the use of a tetrazolium salt procedure, and xanthine oxidase activity was localized with a cerium-diaminobenzidine-cobalt-hydrogen peroxide technique in unfixed cryostat sections of the livers. Cytophotometric measurements showed that total xanthine oxidoreductase activity was decreased after fasting and ischemia, whereas only ischemia caused reduced xanthine oxidase activity. Moreover, the percentage of xanthine oxidase of total xanthine oxidoreductase activity was constant in both periportal and pericentral areas at the level of approximately 4% in normally fed and 24-hour fasted and diethylmaleate-treated rats. Ischemia reduced this percentage in both areas of the liver to 2%. It was concluded that the amount of endogenous reduced glutathione did not affect the percentage of xanthine oxidase. The low percentage of xanthine oxidase as determined in the present in situ histochemical study indicates that in vivo the percentage oxidase in rat liver is lower than is assumed on the basis of biochemical assays in liver homogenates even after strictly controlled homogenization procedures. Apparently, conversion of xanthine dehydrogenase into xanthine oxidase may occur in vitro to yield percentages of xanthine oxidase of 10%-20% as are reported in the literature. The latter increase in the percentage of xanthine oxidase may be caused by changes in the local environment of the enzymes, which is left completely intact in histochemical assays. The finding of this low percentage of xanthine oxidase further stresses that the main function of xanthine oxidoreductase in the liver is not the production of superoxide anion radicals and/or hydrogen peroxide but rather the metabolism of xanthine to uric acid, which can act as a potent antioxidant.

Animals↗
PubMed1995

Regulation of alternative oxidase activity in higher plants.

Plant mitochondria contain two terminal oxidases: cytochrome oxidase and the cyanide-insensitive alternative oxidase. Electron partioning between the two pathways is regulated by the redox poise of the ubiquinone pool and the activation state of the alternative oxidase. The alternative oxidase appears to exist as a dimer which is active in the reduced, noncovalently linked form and inactive when in the oxidized, covalently linked form. Reduction of the oxidase in isolated tobacco mitochondria occurs upon oxidation of isocitrate or malate and may be mediated by matrix NAD(P)H. The activity of the reduced oxidase is governed by certain other organic acids, notably pyruvate, which appear to interact directly with the enzyme. Pyruvate alters the interaction between the alternative oxidase and ubiquinol so that the oxidase becomes active at much lower levels of ubiquinol and competes with the cytochrome pathway for electrons. These requirements for activation of the alternative oxidase constitute a sophisticated feed-forward control mechanism which determines the extent to which electrons are directed away from the energy-conserving cytochrome pathway to the non-energy conserving alternative oxidase. Such a mechanism fits well with the proposed role of the alternative oxidase as a protective enzyme which prevents over-reduction of the cytochrome chain and fermentation of accumulated pyruvate.

Electron Transport Complex IV↗
PubMed2005

NADPH oxidase mediates interleukin-6 expression in cerulein-stimulated pancreatic acinar cells.

NADPH oxidase produces a large amount of reactive oxygen species (ROS) mainly in phagocytic cells. ROS are involved in NF-kappaB activation, cytokine expression and thus, pathogenesis of pancreatitis. However, the source of ROS in pancreatic acinar cells has not been clarified. Cerulein rapidly induces acute and edematous form of pancreatitis. We investigated whether pancreatic acinar cells contain NADPH oxidase, and whether NADPH oxidase mediates interleukin-6 (IL-6) in pancreatic acinar AR42J cells stimulated with cerulein. Expression of NADPH oxidase subunits and NADPH oxidase activity were determined in the cells by immunofluorescence staining and lucigenin luminescence, respectively. Oxidant-sensitive nuclear transcription factor NF-kappaB activation was monitored by electrophoretic mobility shift assay. IL-6 expression was determined by reverse transcription-polymerase chain reaction and enzyme-linked immunosorbant assay. NADPH oxidase inhibitor diphenylene iodonium (DPI), antioxidant rebamipide, and antisense oligonucleotides (AS ODNs) for NADPH oxidase subunits p22phox and p47phox were used to determine the involvement of NADPH oxidase in NF-kappaB activation and IL-6 expression in AR42J cells. As a result, pancreatic acinar AR42J cells constitutively express NADPH oxidase subunits p67phox and p47phox in the cytosol and Nox1 and p22phox in the membrane. Cerulein-stimulated NADPH oxidase activity and induced NF-kappaB activation and IL-6 expression in AR42J cells. Treatment of DPI or rebamipide and transfection of AS ODNs for NADPH oxidase subunits suppressed cerulein-induced NF-kappaB activation and IL-6 expression compared to S ODNs. In conclusion, NADPH oxidase may mediate the expression of inflammatory cytokines by stimulating NF-kappaB activation in pancreatic acinar cells during the course of pancreatitis.

Animals↗
PubMed2004

Lysyl oxidase expression in a rat model of arterial balloon injury.

OBJECTIVE: Traditional therapies for arteriosclerotic disease often fail as a result of an exaggerated fibroproliferative response (recurrent stenosis) at the site of the intervention. Lysyl oxidase, secreted by activated vascular smooth muscle cells and fibroblasts, catalyzes a key step in the cross-linking and stabilization of collagen and elastin in the vascular wall. We hypothesized that lysyl oxidase messenger RNA (mRNA) and protein expression are time-dependent and precede collagen accumulation and luminal narrowing after arterial balloon injury in the rat. METHODS: A 2F balloon-tipped catheter was used to injure the right common carotid artery in male Sprague-Dawley rats. Injured right and control (uninjured) left common carotid arteries were harvested at 0, 0.25, 1, 3, 7, 14, 21, 28, and 60 days for mRNA quantitation and immunohistochemical analysis. Steady-state lysyl oxidase mRNA levels were quantitated with real-time reverse transcription polymerase chain reaction (TaqMan). Immunohistochemical staining with antibodies to alpha-smooth muscle cell actin and lysyl oxidase, and Movat pentachrome staining were performed for qualitative assessment of changes in the cellular and extracellular matrix components of the vessel wall. Post-injury intimal area was measured from hematoxylin and eosin-stained specimens at each time point. RESULTS: When compared with sham-operated control arteries, lysyl oxidase expression in balloon-injured arteries increased significantly to 212% by day 3 after injury, and remained elevated through day 21, with a decrease toward baseline levels by day 28. Lysyl oxidase protein expression did not peak until day 14, and persisted through day 28. Collagen accumulation peaked at day 28, corresponding to the maximal increase in intimal area, with later accumulation of proteoglycans and ground substance in the intimal lesion. CONCLUSION: Our results indicate that lysyl oxidase mRNA and protein expression is time-dependent after balloon injury of the rat carotid artery and that expression appears to precede maximal collagen accumulation and corresponding increases in intimal area. This suggests that lysyl oxidase may have an important role in stabilization of collagen and elastin at sites of vascular injury and that modulation of lysyl oxidase activity may be a viable method to prevent or reduce recurrent stenosis. CLINICAL RELEVANCE: Failure of traditional therapies for ischemic arteriosclerotic disease is often due to an exaggerated fibroproliferative response (recurrent stenosis) at the site of intervention. Recurrent stenosis can be viewed as an injury-repair process, with an initial stage characterized by cellular proliferation followed by deposition of extracellular matrix. This study focuses on lysyl oxidase, a key enzyme involved in stabilization of collagen and elastin. This study demonstrates that lysyl oxidase messenger RNA and protein expression are time-dependent, preceding collagen accumulation and corresponding increases in intimal area. Accumulation of extracellular matrix is a major factor in growth of the restenotic lesion, and modulation of lysyl oxidase activity may offer a therapeutic method for decreasing or preventing recurrent stenosis.

Angioplasty, Balloon↗
PubMed1997

Incorporation of copper into lysyl oxidase.

Lysyl oxidase is a copper-dependent enzyme involved in extracellular processing of collagens and elastin. Although it is known that copper is essential for the functional activity of the enzyme, there is little information on the incorporation of copper. In the present study we examined the insertion of copper into lysyl oxidase using 67Cu in cell-free transcription/translation assays and in normal skin fibroblast culture systems. When a full-length lysyl oxidase cDNA was used as a template for transcription/translation reactions in vitro, unprocessed prolysyl oxidase appeared to bind copper. To examine further the post-translational incorporation of copper into lysyl oxidase, confluent skin fibroblasts were incubated with inhibitors of protein synthesis (cycloheximide, 10 microg/ml), glycosylation (tunicamycin, 10 microg/ml), protein secretion (brefeldin A, 10 microg/ml) and prolysyl oxidase processing (procollagen C-peptidase inhibitor, 2.5 microg/ml) together with 300 microCi of carrier-free 67Cu. It was observed that protein synthesis was a prerequisite for copper incorporation, but inhibition of glycosylation by tunicamycin did not affect the secretion of 67Cu as lysyl oxidase. Brefeldin A inhibited the secretion of 67Ci-labelled lysyl oxidase by 46%, but the intracellular incorporation of copper into lysyl oxidase was not affected. In addition, the inhibition of the extracellular proteolytic processing of prolysyl oxidase to lysyl oxidase had minimal effects on the secretion of protein-bound 67Cu. Our results indicate that, similar to caeruloplasmin processing [Sato and Gitlin (1991) J. Biol. Chem. 266, 5128-5134], copper is inserted into prolysyl oxidase independently of glycosylation.

Bone Morphogenetic Protein 1↗
PubMed1990

Two cytosolic components of the human neutrophil respiratory burst oxidase translocate to the plasma membrane during cell activation.

The superoxide-forming respiratory burst oxidase of human neutrophils is composed of membrane-associated catalytic components and cytosolic constituents required for oxidase activation. This study concerns the hypothesis that cytosolic oxidase components translocate to a membrane fraction when neutrophils are stimulated and the oxidase is activated. A polyclonal antiserum that recognizes two discrete cytosolic oxidase components of 47 and 67 kD was used to probe transfer blots of electrophoresed membrane and cytosol fractions of resting and stimulated neutrophils. In contrast to their strictly cytosolic localization in unstimulated cells, both proteins were detected in membrane fractions of neutrophils activated by phorbol esters and other stimuli. This translocation event was a function of stimulus concentration as well as time and temperature of exposure to the stimulus. It was inhibited by concentrations of N-ethylmaleimide that blocked superoxide formation but was unaffected by 2-deoxyglucose. There was a correlation between translocation of the cytosolic proteins and activation of the oxidase as determined by superoxide formation. Quantitative analyses suggested that approximately 10% of total cellular p47 and p67 became membrane-associated during phorbol ester activation of the oxidase. Analysis of Percoll density gradient fractions indicated that the target membrane for translocation of both proteins was the plasma membrane rather than membranes of either specific or azurophilic granules. In the cell-free oxidase system arachidonate-dependent but membrane-independent precipitation of the cytosolic oxidase proteins was demonstrated. The data show that activation of the respiratory burst oxidase in stimulated human neutrophils is closely associated with translocation of the 47- and 67-kD cytosolic oxidase components to the plasma membrane. We suggest that this translocation event is important in oxidase activation.

Biological Transport↗
PubMed1986

Immunochemical evidence for an inactive form of cytochrome oxidase in mitochondrial membranes of ethanol-fed rats.

Previous studies have established that rats fed ethanol chronically exhibit a 50% decrease in hepatic mitochondrial cytochrome oxidase compared to pair-fed controls, based on both heme aa3 content and specific activity. To determine whether the 'missing' 50% of cytochrome oxidase is present in the membrane but catalytically inactive, or entirely absent, we used immunochemical techniques to determine the content of cytochrome oxidase protein in hepatic submitochondrial particles. Rabbit antiserum against purified rat liver cytochrome oxidase precipitated cytochrome oxidase from detergent-solubilized submitochondrial particles. Immunoinhibition titrations of a fixed amount of anti-oxidase serum with increasing amounts of submitochondrial particle protein showed that similar percentages of added oxidase activity were recovered in supernatants after immunoprecipitation with preparations from both alcoholic and control rats. Similarly, titrations of a fixed amount of submitochondrial particle protein with increasing amounts of antiserum showed comparable decreases in oxidase activity. Equivalent amounts of protein were obtained in immunoprecipitates from both preparations. Immunoprecipitates demonstrated comparable oxidase subunit profiles by electrophoresis, except that one additional band, migrating in the region of oxidase subunit IV, was present in samples from alcoholic rats. The data indicate that cytochrome oxidase immunologic reactivity is quantitatively similar in both types of membranes. The results suggest that the 'missing' cytochrome oxidase is actually present within the membranes of alcoholic animals in an inactive form, apparently devoid of heme aa3.

Animals↗
PubMed1997

Differential and wound-inducible expression of 1-aminocylopropane-1-carboxylate oxidase genes in sunflower seedlings.

In an effort towards understanding the biochemical properties and physiological functions of 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase homologues, we have isolated three ACC oxidase clones from sunflower (Helianthus annuus) seedlings. ACCO1 is a cDNA clone while ACCO2 and ACCO3 and reverse transcriptase-polymerase chain reaction clones. Southern analysis indicated the existence of at least three members in the sunflower ACC oxidase gene family. Expression studies showed that ACCO3 was equally expressed in leaves, hypocotyl, and roots of sunflower seedlings, but it constituted only a small amount of the total ACC oxidase transcripts. In contrast, ACCO1 and ACCO2 were differentially expressed in these organs. ACCO1 mRNA was most abundant in roots, whereas ACCO2 was the major homologue in leaves and in hypocotyl. The levels of total ACC oxidase transcripts in these organs were also determined. High ACC oxidase transcript levels were associated with tissue containing rapidly dividing cells. Wounding and silver ion treatments of hypocotyls increased ACC oxidase mRNA levels and ACC oxidase activity; these events being consistent with the increases in ethylene production. In contrast, ACC oxidase protein levels were not affected by these treatments, suggesting that either a translational regulation and/or a rapid turn-over of the protein is involved in both wound- and silver ion-induced gene expression. Contrary to data in the literature, we found that auxins, ethephon and ACC did not affect ACC oxidase mRNA levels in sunflower hypocotyls. The complexity of ACC oxidase regulation and the significance of organ differential expression of ACC oxidase genes are discussed.

Amino Acid Oxidoreductases↗
PubMed2003

Effect of mutations at Glu160 and Val198 on the thermostability of lactate oxidase.

We have obtained two types of thermostable mutant lactate oxidase - one that exhibited an E-to-G point mutation at position 160 (E160G) through error-prone PCR-based random mutagenesis, and another that exhibited an E-to-G mutation at position 160 and a V-to-I mutation at position 198 (E160G/V198I) through DNA shuffling-based random mutagenesis - both of which we have previously reported. Our molecular modeling of lactate oxidase suggests that the substitution of G for E at position 160 reduces the electrostatic repulsion between the negative charges of E160 and E130 in the (beta/alpha)8 barrel structure, but a thermal-inactivation experiment on the five kinds of single-mutant lactate oxidase at position 160 (E160A, E160Q, E160H, E160R, and E160K) showed that the side-chain volume of the amino acid at position 160 mainly contributes to the thermostability of lactate oxidase. We also produced V198I single-mutant lactate oxidase through site-directed mutagenesis, and analysed the thermostability of wild-type, V198I, E160G, and E160G/V198I lactate oxidase enzymes. The half-life of E160G/V198I lactate oxidase at 70 degrees C was about three times longer than that of E160G lactate oxidase, and was about 20 times longer than that of wild-type lactate oxidase. In contrast, the thermostability of the V198I lactate oxidase was almost identical to that of wild-type lactate oxidase. This indicates that the V198I mutation alone does not affect lactate oxidase thermostability, but does affect it when combined with the E160G mutation.

Amino Acid Sequence↗
PubMed1992

Methanol and ethanol oxidase respiratory chains of the methylotrophic acetic acid bacterium, Acetobacter methanolicus.

Acetobacter methanolicus is a unique acetic acid bacterium which has a methanol oxidase respiratory chain, as seen in methylotrophs, in addition to its ethanol oxidase respiratory chain. In this study, the relationship between methanol and ethanol oxidase respiratory chains was investigated. The organism is able to grow by oxidizing several carbon sources, including methanol, glycerol, and glucose. Cells grown on methanol exhibited a high methanol-oxidizing activity and contained large amounts of methanol dehydrogenase and soluble cytochromes c. Cells grown on glycerol showed higher oxygen uptake rate and dehydrogenase activity with ethanol but little methanol-oxidizing activity. Furthermore, two different terminal oxidases, cytochrome c and ubiquinol oxidases, have been shown to be involved in the respiratory chain; cytochrome c oxidase predominates in cells grown on methanol while ubiquinol oxidase predominates in cells grown on glycerol. Both terminal oxidases could be solubilized from the membranes and separated from each other. The cytochrome c oxidase and the ubiquinol oxidase have been shown to be a cytochrome co and a cytochrome bo, respectively. Methanol-oxidizing activity was diminished by several treatments that disrupt the integrity of the cells. The activity of the intact cells was inhibited with NaCl and/or EDTA, which disturbed the interaction between methanol dehydrogenase and cytochrome c. Ethanol-oxidizing activity in the membranes was inhibited with 2-heptyl-4-hydroxyquinoline N-oxide, which inhibited ubiquinol oxidase but not cytochrome c oxidase. Alcohol dehydrogenase has been purified from the membranes of glycerol-grown cells and shown to reduce ubiquinone-10 as well as a short side-chain homologue in detergent solution.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetobacter↗
PubMed1991

Isoforms of human cytochrome-c oxidase. Subunit composition and steady-state kinetic properties.

The subunit pattern and the steady-state kinetics of cytochrome-c oxidase from human heart, muscle, kidney and liver were investigated. Polyacrylamide gel electrophoresis of immunopurified cytochrome-c oxidase preparations suggest that isoforms of subunit VIa exist, which show differences in staining intensity and electrophoretic mobility. No differences in subunit pattern were observed between the other nucleus-encoded subunits of the various cytochrome-c oxidase preparations. Tissue homogenates, in which cytochrome-c oxidase was solubilised with laurylmaltoside, were directly used in the assays to study the cytochrome-c oxidase steady-state kinetics. Cytochrome-c oxidase concentrations were determined by immunopurification followed by separation and densitometric analysis of subunit IV. When studied in a medium of low ionic strength, the biphasic kinetics of the steady-state reaction between human ferrocytochrome c and the four human cytochrome-c oxidase preparations revealed large differences for the low-affinity TNmax (maximal turnover number) value, ranging from 77 s-1 for kidney to 273 s-1 for liver cytochrome-c oxidase at pH 7.4, I = 18 mM. It is proposed that the low-affinity kinetic phase reflects an internal electron-transfer step. For the steady-state reaction of human heart cytochrome-c oxidase with human cytochrome c, Km and TNmax values of 9 microM and 114 s-1 were found, respectively, at high ionic strength (I = 200 mM, pH 7.4). Only minor differences were observed in the steady-state activity of the various human cytochrome-c oxidases. The interaction between human cytochrome-c oxidase and human cytochrome-c proved to be highly specific. At high ionic strength, a large decrease in steady-state activity was observed when reduced horse, rat or bovine cytochrome c was used as substrate. Both the steady-state TNmax and Km parameters were strongly affected by the type of cytochrome c used. Our findings emphasize the importance of using human cytochrome c in kinetic assays performed with tissues from patients with a suspected cytochrome-c oxidase deficiency.

Animals↗
PubMed1995

New inhibitors of the ubiquinol oxidase of higher plant mitochondria.

A screen has been performed of possible inhibitors of the ubiquinol oxidase of higher plant mitochondria by assaying their effects on cyanide-insensitive NADH oxidase of mitochondria of Arum maculatum. A number of compounds which have powerful inhibitory effects have been identified. Potent inhibition was found with compounds related to the previously described n-propyl gallate, but with the n-propyl sidechain replaced with alkyl chains of greater hydrophobicity. Titration of a range of partial reactions showed that the inhibitors act specifically on the ubiquinol oxidase. The concentrations of inhibitor required are dependent on the respiratory substrate and on the amount of mitochondria used in the assay. Octyl gallate also proved to be a potent inhibitor of the ubiquinol oxidase in tobacco cell suspensions. A second class of compounds which strongly inhibit cyanide-insensitive NADH oxidation is aurachin C and its analogues. Compounds related to aurachin D are much less effective. Titrations of a range of partial reactions indicate that inhibition is caused by a direct action on the ubiquinol oxidase. However, both types of aurachins also act strongly at the Qi site of the cytochrome bc1 complex, as already known to be the case in other systems, and so they are of more limited value for studies of the ubiquinol oxidase. Titration of the oxidation of NADH via the ubiquinol oxidase in a purified mitochondrial fraction from the spadices of Arum maculatum with octyl gallate gave a half-maximal effect at a concentration of around 6 nM when the protein concentration was 14 micrograms ml-1. A similar titre was obtained with a decyl derivative of aurachin C. This allowed us to estimate an upper limit for the concentration of ubiquinol oxidase in these mitochondria of 0.72 +/- 0.15 nmol mg-1 protein, or a ratio of ubiquinol oxidase/cytochrome oxidase of about 15 +/- 7:1. The measurements also provide a minimal turnover number for the ubiquinol oxidase of 186 +/- 42 electrons.s-1. Titration of the ubiquinol oxidase in soybean cotyledon mitochondria with these compounds gave the concentration of inhibitor required to elicit 50% of the maximum observed effect (I50) values about one order of magnitude higher than those found with Arum mitochondria, and again the values depended on the respiratory substrate. An explanation for the variation in I50 values may be found in terms of differences in oxidase concentrations in the different mitochondrial membranes and in the differences in rate-controlling steps with substrates of different activities.

Electron Transport Complex III↗
PubMed1992

Determination of the ligands of the low spin heme of the cytochrome o ubiquinol oxidase complex using site-directed mutagenesis.

The cytochrome o complex of Escherichia coli is a ubiquinol oxidase which is the predominant respiratory terminal oxidase when the bacteria are grown under high oxygen tension. The amino acid sequences of three of the subunits of this quinol oxidase reveal a substantial relationship to the aa3-type cytochrome c oxidases. The two cytochrome components (b563.5 and o) and the single copper (CuB) present in the E. coli quinol oxidase appear to be equivalent to cytochrome a, cytochrome a3, and CuB of the aa3-type cytochrome c oxidases, respectively. These three prosthetic groups are all located within subunit I of the oxidase. Sequence alignments indicate only six totally conserved histidine residues among all known sequences of subunit I of the cytochrome c oxidases of various species plus the E. coli quinol oxidase. Site-directed mutagenesis has been used to change each of these totally conserved histidines with the presumption that two of these six must ligate to the low spin cytochrome center of the E. coli oxidase. The presence of the low spin cytochrome b563.5 component of the oxidase can be evaluated both by visible absorbance properties and by its EPR spectrum. The results unambiguously indicate that His-106 and His-421 are the ligands of the six-coordinate low spin cytochrome b563.5. Although the data are not definitive in making additional metal ligation assignments of the remaining four totally conserved histidines, a reasonable model is suggested for the structure of the catalytic core of the cytochrome o complex and, by extrapolation, of cytochrome c oxidase.

Amino Acid Sequence↗
PubMed1985

Isolation of ubiquinol oxidase from Paracoccus denitrificans and resolution into cytochrome bc1 and cytochrome c-aa3 complexes.

An enzyme complex with ubiquinol-cytochrome c oxidoreductase, cytochrome c oxidase, and ubiquinol oxidase activities was purified from a detergent extract of the plasma membrane of aerobically grown Paracoccus denitrificans. This ubiquinol oxidase consists of seven polypeptides and contains two b cytochromes, cytochrome c1, cytochrome aa3, and a previously unreported c-type cytochrome. This c-type cytochrome has an apparent Mr of 22,000 and an alpha absorption maximum at 552 nm. Retention of this c cytochrome through purification presumably accounts for the independence of ubiquinol oxidase activity on added cytochrome c. Ubiquinol oxidase can be separated into a 3-subunit bc1 complex, a 3-subunit c-aa3 complex, and a 57-kDa polypeptide. This, together with detection of covalently bound heme and published molecular weights of cytochrome c1 and the subunits of cytochrome c oxidase, allows tentative identification of most of the subunits of ubiquinol oxidase with the prosthetic groups present. Ubiquinol oxidase contains cytochromes corresponding to those of the mitochondrial bc1 complex, cytochrome c oxidase complex, and a bound cytochrome c. Ubiquinol-cytochrome c oxidoreductase activity of the complex is inhibited by inhibitors of the mitochondrial bc1 complex. Thus it seems likely that the pathway of electron transfer through the bc1 complex of ubiquinol oxidase is similar to that through the mitochondrial bc1 complex. The number of polypeptides present is less than half the number in the corresponding mitochondrial complexes. This structural simplicity may make ubiquinol oxidase from P. denitrificans a useful system with which to study the mechanisms of electron transfer and energy transduction in the bc1 and cytochrome c oxidase sections of the respiratory chain.

Cell Membrane↗
PubMed1993

The CoA esters of 2-methyl-branched chain fatty acids and of the bile acid intermediates di- and trihydroxycoprostanic acids are oxidized by one single peroxisomal branched chain acyl-CoA oxidase in human liver and kidney.

Rat liver peroxisomes contain three acyl-CoA oxidases: palmitoyl-CoA oxidase, which oxidizes the CoA esters of straight chain fatty acids and prostaglandins; pristanoyl-CoA oxidase, which oxidizes the CoA esters of 2-methyl-branched fatty acids (e.g. pristanic acid); and trihydroxycoprostanoyl-CoA oxidase, which oxidizes the CoA esters of the bile acid intermediates di- and trihydroxycoprostanic acids (Van Veldhoven, P. P., Vanhove, G., Asselberghs, S., Eyssen, H. J., and Mannaerts, G. P. (1992) J. Biol. Chem. 267, 20065-20074). In the present report we demonstrate that human liver peroxisomes contain only two acyl-CoA oxidases: palmitoyl-CoA oxidase, which oxidizes the CoA esters of straight chain fatty acids and prostaglandins, and a novel branched chain acyl-CoA oxidase, which oxidizes the CoA esters of 2-methyl-branched fatty acids as well as those of the bile acid intermediates (which also possess a 2-methyl substitution in their side chains). The branched chain acyl-CoA oxidase was purified to near homogeneity by means of column chromatography. It appeared to be a 70-kDa monomeric protein that did not cross-react with antisera raised against rat palmitoyl-CoA oxidase and pristanoyl-CoA oxidase. No indication was found for the presence of a separate trihydroxycoprostanoyl-CoA oxidase in human liver. The branched chain acyl-CoA oxidase was present also in human kidney, suggesting that it is expressed in other extrahepatic tissues as well. Our results explain a number of clinical-chemical observations made in certain cases of peroxisomal beta-oxidation disorders.

Acyl Coenzyme A↗