Purification, cDNA Cloning, and Tissue Distribution of Bovine Liver Aldehyde Oxidase

Molecular characterization of male and female rat liver aldehyde oxidase is reported. As described for the mouse liver, male and female rat liver expressed kinetically distinct forms of aldehyde oxidase. Our data suggest that the two forms arise as a result of differences in redox state and are most simply explained by expression of a single gene encoding aldehyde oxidase in rats. In support of this argument we have sequenced cDNAs from male and female rat liver. We examined mRNA expression by Northern blot analysis with RNA from males and females, from several tissues, and following androgen induction. Purified rat liver enzyme from males or females revealed a single 150-kDa species consistent with cDNA sequence analysis. Both male and female forms were reactive to the same carboxyl-terminal directed antisera. K m(app) values obtained in crude extracts of male or female rat liver and post-benzamidine-purified aldehyde oxidase differed substantially from each other but could be interconverted by chemical reduction with dithiothreitol or oxidation with 4,4-dithiodipyridine. Our data indicate that a single gene is most likely expressed in male or female rat liver and that the kinetic differences between male and female rat liver aldehyde oxidases are sensitive to redox manipulation. Aldehyde oxidase (AOX)1 is a member of the molybdenum cofactor containing enzymes. Native AOX is routinely prepared as a homodimer of 300 kDa. Each 150-kDa subunit contains two iron-sulfur centers, an FAD, and the molybdenum-pterin cofactor (MoCo) (1-3). AOX (EC 1.2.3.1) catalyzes the oxidation of a wide range of aldehydic compounds, purines, quinoliniums, and numerous pharmacologic agents. While substrate specificity for AOX is very broad, and wide species variation in substrate specificity exists, the general catalytic reaction takes the form of hydroxyl transfer from water to an aldehyde creating the cognate acid. For example, conversion of benzaldehyde to benzoic acid is a very efficient reaction for AOX from most species. Conversion of N-1-methyl nicotinamide (NMN) to the 2-or -4-pyridone has been used as a standard definition of AOXs, although it is usually a kinetically less efficient reaction than benzaldehyde oxidation.AOX is of interest both for its role in drug metabolism and as a source of the reactive oxygen species (ROS), hydrogen peroxide, and the superoxide anion, that have been related to numerous human pathologies. The human gene encoding AOX has been linked to a rare form of amyotrophic lateral sclerosis, although it is unknown if AOX encodes the amyotrophic lateral sclerosis locus itself (4 -6). ROS are generated from AOX in an oxidative half-reaction following reduction of the enzyme by substrate. The FeS, FAD, and MoCo cofactors comprise an internal electron transfer chain in which electrons are passed from the active site molybdenum center to the FeS centers and finally to FAD. Partial reduction of oxygen at the the reduced FAD site produces ROS. Unlike the related enzyme xanthine dehydrogenase (XDH), AOX does no...

Section: Table II Sequence Differences In Male and Female Rat Aox1mentioning

confidence: 99%

mentioning

confidence: 99%

cDNA Cloning, Sequencing, and Characterization of Male and Female Rat Liver Aldehyde Oxidase (rAOX1)

Wright

Clayton

Riley

et al. 1999

“…Partial Purification of Mouse Liver AOH1 and Column Chromatography-The AOH1 protein was partially purified from mouse liver by benzamidine-Sepharose affinity chromatography (Amersham Pharmacia Biotech) exactly as described for bovine AO (13). Following elution with benzamidine (Sigma), active fractions were concentrated by ultrafiltration on Centricon YM-100 cartridges (Millipore, Bedford, MA) and applied to a gel filtration FPLC Superose-6 column (Amersham Pharmacia Biotech) run at 0.5 ml/min in phosphate-buffered saline (pH 7.4), containing 100 mM NaCl.…”

Section: Molecular Cloning and Sequencing Of The Cdnas Encoding Aoh1mentioning

confidence: 99%

“…In mammals, three molybdo-proteins have been identified and characterized, i.e. sulfite oxidase (7), xanthine oxidoreductase (XOR), 1 (8 -12) and aldehyde oxidase (AO) (13)(14)(15)(16).…”

mentioning

confidence: 99%

Cloning of the cDNAs Coding for Two Novel Molybdo-flavoproteins Showing High Similarity with Aldehyde Oxidase and Xanthine Oxidoreductase

Terao

Kurosaki

Saltini

et al. 2000

Self Cite

113

The cDNAs coding for two novel mouse molybdo-flavoproteins, AOH1 and AOH2 (aldehyde oxidase homolog 1 and 2), were isolated. The AOH1 and AOH2 cDNAs code for polypeptides of 1336 amino acids. The two proteins have similar primary structure and show striking amino acid identity with aldehyde oxidase and xanthine oxidoreductase, two other molybdo-flavoenzymes. AOH1 and AOH2 contain consensus sequences for a molybdopterin-binding site and two distinct 2Fe-2S redox centers. In its native conformation, AOH1 has a molecular weight consistent with a homotetrameric structure. Transfection of the AOH1 and AOH2 cDNAs results in the production of proteins with phenanthridine but not hypoxanthine oxidizing activity. Furthermore, the AOH1 protein has benzaldehyde oxidizing activity with electrophoretic characteristics identical to those of a previously identified aldehyde oxidase isoenzyme (Holmes, R. S. (1979) Biochem. Genet. 17, 517-528). The AOH1 transcript is expressed in the hepatocytes of the adult and fetal liver and in spermatogonia. In liver, the AOH1 protein is synthesized in a gender-specific fashion. The expression of AOH2 is limited to keratinized epithelia and the basal layer of the epidermis and hair folliculi. The selective cell and tissue distribution of AOH1 and AOH2 mRNAs is consistent with the localization of the respective protein products.

“…This has focused our attention on the development of efficient methods for the purification of the enzymes from their native sources (7,13,14). Comparison of the enzymatic properties of AOX1 and AOH1 is particularly relevant and difficult.…”

mentioning

confidence: 99%

Regulation and Biochemistry of Mouse Molybdo-flavoenzymes

Vila¹,

Kurosaki²,

Barzago³

et al. 2004

Self Cite

Mouse molybdo-flavoenzymes consist of xanthine oxidoreductase, aldehyde oxidase (AOX1), and two recently identified proteins, AOH1 and AOH2 (aldehyde oxidase homologues 1 and 2). Here we demonstrate that CD-1, C57BL/6, 129/Sv, and other mouse strains synthesize high levels of AOH1 in the liver and AOH2 in the skin. By contrast, the DBA/2 and CBA strains are unique, having a selective deficit in the expression of the AOH1 and AOH2 genes. DBA/2 animals synthesize trace amounts of a catalytically active AOH1 protein. However, relative to CD-1 animals, an over 2 log reduction in the steady-state levels of liver AOH1 mRNA, protein, and enzymatic activity is observed in basal conditions and following administration of testosterone. The DBA/2 mouse represents a unique opportunity to purify AOX1 and compare its enzymatic characteristics to those of the AOH1 protein. The spectroscopy and biochemistry of AOX1 are very similar to those of AOH1 except for a differential sensitivity to the non-competitive inhibitory effect of norharmane. AOX1 and AOH1 oxidize an overlapping set of aldehydes and heterocycles. For most compounds, the substrate efficiency (V max /K m ) of AOX1 is superior to that of AOH1. Alkylic alcohols and acetaldehyde, the toxic metabolite of ethanol, are poor substrates of both enzymes. Consistent with this, the levels of acetaldehyde in the livers of ethanol administered CD-1 and DBA/2 mice are similar, indicating that neither enzyme is involved in the in vivo biotransformation of acetaldehyde.