Aldehyde Oxidase Activity Research Articles

Sulphoxide reduction by rat and rabbit tissues in vitro

The reduction of sulindac, sulphinpyrazone and diphenyl sulphoxide to their thioether analogues has been studied in vitro using rat and rabbit tissues. Sulindac reduction was about 10-fold higher in homogenates of rat kidney and liver than in other tissues although the tissue differences decreased when dithiothreitol was used as a co-factor. The greatest sulindac reducing activity in rat liver was in the cytosolic fraction whereas reoxidation of the thioether back to the sulphoxide was largely in the microsomal fraction. Studies using NADPH/NADH, acetaldehyde and dithiothreitol as cofactors showed that aldehyde oxidase was the main sulindac reducing system in rat and rabbit liver cytosols but not in renal cytosols where reduction was probably linked to the thioredoxin system, as reported previously. Menadione and hydralazine caused essentially complete inhibition of sulindac reduction by hepatic but not renal cytosol and the inhibition was dependent on preincubation of the enzyme with the inhibitor, which is indicative of aldehyde oxidase activity. Little reduction of sulphinpyrazone or diphenyl sulphoxide was detected with rat or rabbit kidney or renal cytosols, although increased reduction was detected when acetaldehyde was added as a cofactor to rabbit and rat liver cytosols. The data indicate that different enzyme systems are responsible for sulphoxide reduction in the liver and kidney.

Molybdenum Cofactor Mutants, Specifically Impaired in Xanthine Dehydrogenase Activity and Abscisic Acid Biosynthesis, Simultaneously Overexpress Nitrate Reductase.

The molybdenum cofactor is shared by nitrate reductase (NR), xanthine dehydrogenase (XDH), and abscisic acid (ABA) aldehyde oxidase in higher plants (M. Walker-Simmons, D.A. Kudrna, R.L. Warner [1989] Plant Physiol 90:728-733). In agreement with this, cnx mutants are simultaneously deficient for these three enzyme activities and have physiological characteristics of ABA-deficient plants. In this report we show that aba1 mutants, initially characterized as ABA-deficient mutants, are impaired in both ABA aldehyde oxidase and XDH activity but overexpress NR. These characteristics suggest that aba1 is in fact involved in the last step of molybdenum cofactor biosynthesis specific to XDH and ABA aldehyde oxidase; aba1 probably has the same function as hxB in Aspergillus. The significance of NR overexpression in aba1 mutants is discussed.

Comparison of levels of aldehyde oxidase with cytochrome P450 activities in human liver in vitro

Microsomal suspensions and 9000 g supernatant (S-9) fractions were prepared from the liver tissue of six human multiorgan donors. The S-9 fractions were characterized for cytosolic aldehyde oxidase (AO) activity, using three different substrates [ N 1-methylnicotinamide (NMN), benzaldehyde and 6-methylpurine]. In addition, human liver NMN oxidase activity was compared with that detected in rat, dog and monkey liver S-9 fractions. As expected, the rank order of NMN oxidase activity was monkey > rat > dog, and in five out of six subjects the activity was lowest in humans (<2.0 nmol/ min/ mg). The variation in AO activity among the various human livers was greater for NMN oxidase (>40 fold) than for 6-methylpurine and benzaldehyde oxidase (⩽3.6 fold) activity. The corresponding microsomal preparations were characterized with respect to the levels of total cytochrome P450 (CYP) and six CYP-dependent mixed-function monooxygenase (MFO) activities. The variation was greatest for dextromethorphan O-demethylase (CYP2D6) and lowest for N, N-dimethylnitrosamine N-demethylase (CYP2E1) activity (147- vs 1.4-fold). The inter-sample variation for the total CYP, CYP3A (erythromycin N-demethylase), CYP1A2 (7-ethoxyresorufin O-deethylase), CYP2A6 (coumarin 7-hydroxylase) and CYP2C9/10 (tolbutamide 4-methyl hydroxylase) was 2.2- to 5.3-fold. Furthermore, the levels of AO activity did not correlate with total (spectrally detectable) CYP or any of the CYP form-selective MFO activities.

Effect of sex hormones on lipid peroxidation in rat liver.

The role of sex hormones in hepatic lipid peroxidation, and in hepatic aldehyde oxidase and xanthine oxidase activities were investigated using rat liver homogenates. It was observed that male rat had a significantly greater content of malondialdehyde in liver than female. Among the sex hormones tested, estradiol, one of female hormones, markedly inhibited the formation of lipid peroxides in liver tissues in vitro. Especially, the inhibitory effect of estradiol appeared more remarkably in Fe+2-induced lipid peroxidation. The hepatic xanthine oxidase activity was decreased about 15% by 10(-6) M estradiol, whereas, the aldehyde oxidase activity was almost completely disappeared at the same concentration of estradiol. It implies that sex differences in lipid peroxidation is attributed to the suppression of free radical generating system by estradiol.

Epoxide reductase activity of mammalian liver cytosols and aldehyde oxidase.

The present study provides the first evidence that a mammalian liver cytosolic enzyme, aldehyde oxidase, has an ability to reduce arene oxides to the parent hydrocarbons under anaerobic conditions. The comparative ability of rabbit liver preparations to reduce arene oxides was examined using naphthalene 1,2-oxide and benzo[a]pyrene 4,5-oxide as substrates. The liver cytosol with an electron donor of aldehyde oxidase exhibited much higher epoxide reductase activity compared with the liver microsomes with NADPH and FAD. The cytosolic activity was sensitive to inhibitors of aldehyde oxidase. Purified rabbit liver aldehyde oxidase also exhibited a significant epoxide reductase activity in the presence of its electron donor. Apparent Km and Vmax values of the enzyme were 426 microM and 323 nmol/min/mg protein for naphthalene 1,2-oxide and 255 microM and 100 nmol/min/mg protein for benzo[a]pyrene 4,5-oxide respectively. However, no epoxide reduction by the enzyme or by the liver cytosol was detected in olefin epoxides such as styrene oxide and trans-stilbene oxide. Similar results were obtained with rat liver preparations. However, the epoxide reductase activity of cytosol and aldehyde oxidase from rat liver was considerably lower than that of the rabbit liver preparations. In hamsters, mice and guinea-pigs, liver cytosols with an electron donor of aldehyde oxidase as well as liver microsomes with NADPH exhibited a significant epoxide reductase activity toward naphthalene 1,2-oxide. However, no epoxide reduction was observed with dog liver cytosol. Administration of sodium tungstate to rats depleted liver cytosolic reductase activity and sodium molybdate treatment resulted in partial restoration of the activity, supporting the view that the epoxide reductase activity observed in the liver cytosol mainly originates from aldehyde oxidase.

Effect of BOF-4272 on the oxidation of allopurinol and pyrazinamide in vivo: Is xanthine dehydrogenase or aldehyde oxidase more important in oxidizing both allopurinol and pyrazinamide?

Allopurinol or pyrazinamide was administered to rats treated with BOF-4272 (a potent xanthine oxidase inhibitor) to investigate to what degree xanthine dehydrogenase participates in the oxidation of these agents. BOF-4272 markedly decreased the plasma concentration and the urinary excretion of both oxypurinol and 5-hydroxypyrazinamide. It also decreased the sum of the urinary excretion of allopurinol and oxypurinol and that of pyrazinamide and its metabolites, although it did not affect the sum of the plasma concentrations of allopurinol and oxypurinol at 105 min after administration of allopurinol or the plasma concentration of pyrazinamide during the period after the administration of pyrazinamide. These results suggested that BOF-4272 almost completely inhibited the oxidation of allopurinol and pyrazinamide and had some effect on the excretion and/or the tissue incorporation of these two compounds. Since the in vitro study demonstrated that BOF-4272 did not inhibit the activity of aldehyde oxidase, which oxidazed both allopurinol to oxypurinol and pyrazinamide to 5-hydroxyprazinamide, the results suggested that xanthine dehydrogenase was the more important enzyme in converting allopurinol to oxypurinol and pyrazinamide to 5-hydroxypyrazinamide.

Aromatic alcohol oxidase and aldehyde oxidase activities in the digestive gland of three species of terrestrial gastropod ( Helix aspersa, arion ater, limax flavus)

1. 1. Digestive gland homogenates of three species of terrestrial gastropod mollusc were used to prepare sources of soluble aldehyde oxidase and solubilized aromatic alcohol oxidase activites. 2. 2. The snail ( Helix aspersa) had two aldox isoenzymes each able to utilize salicylaldehyde whereas two slug species had a single apparent activity inhibitable by salicylaldehyde. 3. 3. Aromatic alcohol oxidase activities of the three gastropods differed somewhat in their relative abilities to oxidize various methoxy-benzyl and amino-benzyl alcohols but, unlike fungal aromatic alcohol oxidase, were all able to utilize crotonyl alcohol as substrate.

Subcellular localisation of guinea pig hepatic molybdenum hydroxylases

Molybdenum hydroxylase activity in guinea pig liver has been compared with that of marker enzymes in mitochondria (succinate dehydrogenase), microsomes (glucose-6-phosphatase) and cytosol (lactate dehydrogenase). Aldehyde oxidase activity was highest in the cytosol, with about 10-fold activity of xanthine oxidase. Significant molybdenum hydroxylase activity was found in mitochondria with minimal levels in microsomes. Mitochondrial and cytosolic aldehyde oxidase varied in substrate specificity and electrophoretic mobility with two major bands in each fraction, one of which was common to cytosol and mitochondria.

Abscisic Aldehyde Is an Intermediate in the Enzymatic Conversion of Xanthoxin to Abscisic Acid in Phaseolus vulgaris L. Leaves

The enzymatic conversion of xanthoxin to abscisic acid by cell-free extracts of Phaseolus vulgaris L. leaves has been found to be a two-step reaction catalyzed by two different enzymes. Xanthoxin was first converted to abscisic aldehyde followed by conversion of the latter to abscisic acid. The enzyme activity catalyzing the synthesis of abscisic aldehyde from xanthoxin (xanthoxin oxidase) was present in cell-free leaf extracts from both wild type and the abscisic acid-deficient molybdopterin cofactor mutant, Az34 (nar2a) of Hordeum vulgare L. However, the enzyme activity catalyzing the synthesis of abscisic acid from abscisic aldehyde (abscisic aldehyde oxidase) was present only in extracts of the wild type and no activity could be detected in either turgid or water stressed leaf extracts of the Az34 mutant. Furthermore, the wilty tomato mutants, sitiens and flacca, which do not accumulate abscisic acid in response to water stress, have been shown to lack abscisic aldehyde oxidase activity. When this enzyme fraction was isolated from leaf extracts of P. vulgaris L. and added to extracts prepared from sitiens and flacca, xanthoxin was converted to abscisic acid. Abscisic aldehyde oxidase has been purified about 145-fold from P. vulgaris L. leaves. It exhibited optimum catalytic activity at pH 7.25 in potassium phosphate buffer.

Demonstration of a combined deficiency of xanthine oxidase and aldehyde oxidase in xanthinuric patients not forming oxipurinol

Genetic heterogeneity has been suggested in xanthinuria from the hitherto unexplained ability of some patients with this hereditary disorder to convert allopurinol to its active metabolite oxipurinol--an activity generally attributed to xanthine oxidase. This study provides evidence that the enzyme aldehyde oxidase is also deficient in xanthinuric patients not converting allopurinol to oxipurinol, whereas a xanthinuric patient with normal formation of oxipurinol had normal aldehyde oxidase activity. It is concluded that the enzyme aldehyde oxidase is the principal enzyme responsible for the formation of oxipurinol in man.

Aldehyde oxidases and dehydrogenases in antennae of five moth species

Conversion of Z11–16:A1 to its corresponding carboxylic acid was examined in male antennal extracts from Heliothis subflexa, Heliothis virescens, Heliothis zea, Manduca sexta and Spodoptera frugiperda moths. Both aldehyde dehydrogenase (ALDH) and aldehyde oxidase (AO) activities were detected by radiochromatographic assays using [ 3H]Z11–16:A1 as the substrate and by spectroscopic assays using Z11–16:A1 as the substrate. AO activity in each of the five moths showed a broad substrate specificity which included C 4 to C 18 aliphatic aldehydes and benzaldehyde. ALDH and AO enzymes from the male and female antennal extracts were identified after electrophoretic protein separation. AO enzymes were visualized on native polyacrylamide gels with formazan- or horseradish peroxidase-mediated stain coupled to the AO-catalyzed oxidation of benzaldehyde. Both sexes of most species showed intense AO-stained bands of similar mobility. Molecular subunits of potential ALDH enzymes were visualized by fluorography of SDS-PAGE-separated proteins after covalent modification by [ 3H](Z)-1,11-hexadecadien-3-one, a selective affinity label for ALDH. ALDH subunits appeared at 51 ± 1 kDa in antennae from males of all five species and females of three species; female H. zea and M. sexta moths lacked these characteristic ALDH subunits. These results constitute the first biochemical comparisons of coexisting ALDH and AO enzymes in antennal extracts from lepidopteran species from two families.

Aromatic alcohol oxidase: A new membrane‐bound H2O2‐generating enzyme in alimentary tissues of the slug Arion ater

AbstractWe employed a sensitive spectrophotometric technique to detect H2O2‐generating enzymic activities capable of utilizing aldehydes or alcohols as substrates. We analysed tissue homogenates and subcellular fractions of the terrestrial slug Arion ater. Of the tissues tested, those of the alimentary system (especially crop and digestive gland) had the greatest activity and generated H2O2 when supplied with a variety of alcohol and aldehyde substrates. Aldehyde oxidase activity was soluble and had high apparent affinity for benzaldehyde. This activity is probably analogous to mammalian aldox (aldehyde oxidase; E.C.1.2.3.1), since it could use an artificial electron acceptor system and greatly preferred benzaldehyde to hypoxanthine. However, it differed from guinea pig liver aldehyde oxidase in that salicylaldehyde was a potent inhibitor rather than a substrate. Alcohol oxidase activity was particulate but not localised in mitochondria or peroxisomes. In digestive gland, it was localised differently from mannitol oxidase. In crop, the subcellular sites are either the same or inseparable by isopycnic density gradient centrifugation in sucrose gradients. Activity was at least ten times greater with aromatic alcohols than with ethanol, propanol, or butanol. No activity was detected with methanol, and the enzyme could not utilise an artificial electron acceptor system or pyridine nucleotides. Aromatic alcohol oxidase differed from mannitol oxidase with respect to temperature and pH sensitivity and in exhibiting greater apparent affinity for its substrates. The alcohol oxidase accepted a wide variety of aromatic alcohols as substrates. In several respects it resembled the corresponding enzyme of wood‐rot fungi, implying that the slug utilises dietary lignin or lignin precursors. Possible dietary, microbicidal, and detoxifying roles are considered for slug aldehyde and alcohol oxidases.

Reduced Accumulation of ABA during Water Stress in a Molybdenum Cofactor Mutant of Barley

A barley (Hordeum vulgare L.) mutant (Az34) has been identified with low basal levels of abscisic acid (ABA) and with reduced capacity for producing ABA in response to water stress. The mutation is in a gene controlling the molybdenum cofactor resulting in a pleiotropic deficiency in at least three molybdoenzymes, nitrate reductase, xanthine dehydrogenase, and aldehyde oxidase. The mutant was found to lack aldehyde oxidase activity with several substrates including: (a) ABA aldehyde, a putative precursor of ABA; (b) an acetylenic analog of ABA aldehyde; and (c) heptaldehyde. Elevating the growth temperature from 18 to 26 degrees C caused mutant leaves to wilt and brown. Desiccation of mutant leaves was prevented by applying ABA. These results indicate that ABA biosynthesis at some developmental stages is dependent upon a molybdoenzyme which may be an aldehyde oxidase.

Diurnal variation and melatonin induction of hepatic molybdenum hydroxylase activity in the guinea-pig

The activities of the xenobiotic metabolizing enzymes, aldehyde oxidase and xanthine oxidase, were determined in partially purified fractions of adult guinea-pig liver at given times in the day or night. A marked circadian variation in aldehyde oxidase activity was observed with several substrates (phthalazine, phenanthridine, N-phenylquinolinium and 3, 4-dihydro-4-hydroxy-3-methyl-2-quina-zolinone). The main peak occurred at 0300 hr with minimum activity from 1200 to 1800 hr, the differences between rhythmic extremes being statistically significant (P < 0.005). Xanthine oxidase activity also exhibited a daily rhythm but with a lower amplitude. Guinea-pig serum melatonin showed a synchronous circadian fluctuation with peak values at 0300 hr falling throughout the day to a minimum at 1800 hr. Exogenously administered melatonin caused a significant increase in aldehyde oxidase activity at 0900 and 1200 hr and in xanthine oxidase activity at 0900 hr. It was concluded that melatonin concentrations may be related to the circadian variation in liver molybdenum hydroxylase activity.

Molecular enzymology of the reductive bioactivation of hypoxic cell cytotoxins

The hypoxic cell cytotoxins SR 4233, benznidazole (Benzo), and CB 1954 were readily reduced by anaerobic mouse liver microsomes in vitro to their respective amino or single N-oxide derivatives. The reactions were inhibited in air and required reduced cofactors, particularly NADPH. The rates of reductive bioactivation were markedly different for each drug, with SR 4233 ⪢ CB 1954 > Benzo. Using purified cytochrome P-450 reductase (P-450 reductase) and an inhibitory antibody to this enzyme, we demonstrated that P450 reductase was involved in the reductive bioactivation of all 3 compounds. It had a minor role in SR 4233 reduction, but a more important involvement in CB 1954 metabolism to its 4-amino metabolite. Using carbon monoxide, a specific inhibitor of cytochrome P-450 (P-450~ we demonstrated that P450 was involved in both SR 4233 and Benzo reduction. P450 had a major role both in SR 4233 conversion to SR 4317 and in the latter steps of Benzo amine formation. Purified xanthine oxidase was shown to reduce SR 4233 and Benzo in vitro, but cytosolic aldehyde oxidase activity was only detectable with Benzo as substrate. Characterizing the relative participation of the various reductases in tumor versus normal tissues may allow a more rational selection and application of hypoxic cell cytotoxins in cancer therapy.

Peroxidase as a model for reduction of tertiary amine oxides catalyzed by rat hepatic supernatant and microsomal fractions

Rat hepatic microsomal and 100,000 g supernatant fractions catalyzed an NADH- and FMN-dependent reduction of amine oxides. Horseradish peroxidase (HRP) served as a model for the amine oxide reductase located in rat hepatic 100,000 g supernatant fraction. The HRP-catalyzed reaction displayed saturation kinetics with respect to NADH and the amine oxide substrate; however, there was an optimum concentration for FMN after which inhibition was observed at increased concentrations of FMN. The reductase in the 100,000 g hepatic supernatant fraction closely paralleled HRP-catalyzed amine oxide reduction in coenzyme requirements, sensitivity to inhibitors, and substrate specificity. Moreover, the peroxidase activity of HRP and microsomal and 100,000 g supernatant fractions correlated with the NADH- and FMN-dependent amine oxide reductase activities of these enzyme preparations. The NADH- and FMN-dependent amine oxide reductase activity in 100,000 g supernatant fractions, however, did not parallel the aldehyde oxidase activity. Thus, the results indicate that there is an amine oxide reductase in rat hepatic 100,000 g supernatant fraction with catalytic properties that are modeled well by horseradish peroxidase.

Purification of aldehyde oxidase from bovine ciliary body.

Ocular aldehyde oxidase was purified for the first time from bovine ciliary body cytosol by ammonium sulfate fractionation and successive HPLC using DEAE anion-exchange and hydroxyapatite columns. The purified enzyme was homogeneous by the criterion of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weight of the enzyme was estimated to be about 150,000 by electrophoresis and to be about 300,000 by gel filtration HPLC on a TSK gel G3000SWXL column, indicating that the enzyme consists of two subunits with the same molecular weight. On the other hand, nicotinamide N-oxide reductase activity was associated with aldehyde oxidase activity throughout the purification steps of the latter enzyme. This fact indicated that nicotinamide N-oxide reductase activity of the ciliary body cytosol is due to aldehyde oxidase present in the tissue preparation.

Molybdoenzymes in Drosophila. IV. Further characterization of the cinnamon phenotype

Mutations at the cin gene display drastically lowered levels of the molybdoenzymes, xanthine dehydrogenase (XDH) and aldehyde oxidase (AO), and lack pyridoxal oxidase (PO) and sulfite oxidase (SO) activities. Certain mutations at cin also display varying degrees of female sterility, which is maternally affected. Here we characterize five new cin alleles with respect to the molybdoenzyme activities as well as the molybdenum cofactor, commonly required for molybdoenzyme activity. In complementing cin heterozygotes we find that, in addition to the previously reported unusually high levels of XDH and AO activities, there are unusually elevated levels of SO activity, as well as complementation for PO activity. The levels of immunologically crossreacting material in such heterozygotes indicate that the elevated levels of molybdoenzyme activities cannot be due to increases in the number of enzyme molecules. Measurements of the level of molybdenum cofactor activity normally present in XDH, AO, PO, and SO point to the possibility that a larger fraction of the enzyme molecules are active in these heterozygotes. The possible role of SO with respect to cinnamon's female sterility is also discussed.

Metabolism of drugs in the eye. Drug-reducing activity of preparations from bovine ciliary body.

Drug-metabolizing activities, especially the reductase activities towards N-oxide, hydroxamic acid, sulfoxide and nitro compounds were comparatively examined with bovine ciliary body. As described previously, the cytosol from the ocular tissue exhibits the nicotinamide N-oxide reductase activity when supplemented with 2-hydroxypyrimidine, an electron donor of aldehyde oxidase. When the cytosol was fractionated with ammonium sulfate, followed by assays of aldehyde oxidase and nicotinamide N-oxide reductase activities in each fraction, the distribution of aldehyde oxidase activity in the resultant ammonium sulfate fractions was nearly parallel to that of nicotinamide N-oxide reductase activity. Furthermore, reductase activities towards drugs such as sulfoxide, hydroxamic acid and nitro compounds were observed with the cytosol in the presence of 2-hydroxypyrimidine or N1-methylnicotinamide. In general, these reductase activities of the fraction were markedly inhibited by menadione, an inhibitor of aldehyde oxidase. These results suggest that aldehyde oxidase present in ciliary body plays an important role in the reduction of a variety of xenobiotics in mammalian eyes. However, in the case of imipramine N-oxide, its reduction in the ocular tissue appears to be more readily catalyzed by a menadione-linked enzyme different from aldehyde oxidase.

Species variation in hepatic aldehyde oxidase activity

The activity of hepatic aldehyde oxidase from rabbit, guinea pig, rat, marmoset, dog, baboon and man was investigated in vitro with charged and uncharged N-heterocyclic substrates: Km and Vmax values were determined for phthalazine, 6,7-dimethoxy-1-[-4-(ethylcarbamoyloxy)piperidino]phthalazine (carbazeran), quinine and quinidine. The oxidation of N-phenylquinolinium chloride to N-phenyl-2-quinolone and N-phenyl-4-quinolone was followed spectrophotometrically. Rat or dog liver showed low and negligible enzyme activity respectively, whereas baboon liver contained a highly active aldehyde oxidase. Enzyme from marmoset and guinea pig liver had the closest spectrum of activity to human liver aldehyde oxidase. Unlike that from man, rabbit hepatic aldehyde oxidase was refractory towards carbazeran and converted N-phenylquinolinium chloride predominantly to the 2-quinolone. N-Phenyl-4-quinolone was the major oxidation product with enzyme from guinea pig, marmoset, baboon and man.