Purification of the aldehyde oxidase homolog 1 (AOH1) protein and cloning of the AOH1 and aldehyde oxidase homolog 2 (AOH2) genes. Identification of a novel molybdo-flavoprotein gene cluster on mouse chromosome 1.

We report the cloning of the AOH1 and AOH2 genes, which encode two novel mammalian molybdo-flavoproteins. We have purified the AOH1 protein to homogeneity in its catalytically active form from mouse liver. Twenty tryptic peptides, identified or directly sequenced by mass spectrometry, confirm the primary structure of the polypeptide deduced from the AOH1 gene. The enzyme contains one molecule of FAD, one atom of molybdenum, and four atoms of iron per subunit and shows spectroscopic features similar to those of the prototypic molybdo-flavoprotein xanthine oxidoreductase. The AOH1 and AOH2 genes are 98 and 60 kilobases long, respectively, and consist of 35 coding exons. The AOH1 gene has the potential to transcribe an extra leader non-coding exon, which is located downstream of exon 26, and is transcribed in the opposite orientation relative to all the other exons. AOH1 and AOH2 map to chromosome 1 in close proximity to each other and to the aldehyde oxidase gene, forming a molybdo-flavoenzyme gene cluster. Conservation in the position of exon/intron junctions among the mouse AOH1, AOH2, aldehyde oxidase, and xanthine oxidoreductase loci indicates that these genes are derived from the duplication of an ancestral precursor.

Preventive effect of diethyldithiocarbamate on selenite-induced opacity in cultured rat lenses.

Increasing evidence suggests the involvement of reactive oxygen species in the development of cataracts. In this study, we investigated the preventive effect of diethyldithiocarbamate (DDC) on the selenite-induced opacification of cultured rat lenses. Lens opacity was produced by incubation with 0.2 mM selenite for 24 h, which resulted in an increase in selenium content in the cultured lenses. The increase in selenium content and the onset of opacification and lens membrane damage were inhibited by preincubation with DDC. It is reasonable to assume that DDC contributed to anticataract ability. In addition, selenite resulted in a significant decrease in glutathione and protein thiol content and an increase in lipid peroxidation levels in the lenses. These alterations were also depressed by DDC, suggesting a contribution of an antioxidative effect by DDC in the inhibition of lens opacification. At the same lens selenium content, DDC treatment inhibited opacification and lipid peroxide. In conclusion, we propose that the antioxidative properties of DDC play a major role in its contribution to the anticataract effect.

Cloning of the cDNAs coding for two novel molybdo-flavoproteins showing high similarity with aldehyde oxidase and xanthine oxidoreductase.

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.

Comparative study of superoxide dismutase in normal and hereditary cataract (UPL) rats.

In the present study, the levels of SOD activity and Cu, Zn-SOD mRNA in the brain, kidney, liver and eye of normal and Upjohn Pharmaceutics Limited (UPL) rats, a new hereditary cataract model derived from Sprague-Dawley rats, were measured. Although the levels of SOD activity in the eye and brain of UPL rats were significantly decreased compared with those of normal rats 3 and 5 weeks after birth, the levels of SOD activities in the kidney and liver were the same in both groups. The levels of Cu, Zn-SOD mRNA in kidney and liver of UPL rats were the same as those of normal controls. The level of Cu, Zn-SOD mRNA in the brain of normal rats 5 weeks after birth was about twofold greater than that of UPL, and that in the eye of UPL rats 3 weeks after birth was significantly decreased compared with that of normal controls. The sequences of cDNA encoding Cu, Zn-SOD and the sequences of the regulatory region of the Cu, Zn-SOD gene were confirmed to be the same in normal and UPL rats. These results indicated that the decreases in levels of SOD activity and Cu, Zn-SOD mRNA in the brain and eye of UPL rat were not due to mutation of the genomic Cu, Zn-SOD gene in UPL rats or differences in the sequence of the regulatory region of the Cu, Zn-SOD gene between normal and UPL rats.

Correlation between prevention of cataract development by disulfiram and fates of selenium in selenite-treated rats.

PURPOSE: We found a new pharmacological effect of disulfiram (DSF) against rat pups with cataract induced by selenite injection. The possible reactive mechanism is discussed in this present paper. METHODS: Wistar male and female rats aged 13 and 30 days, and male rats aged 7 weeks were used this present study. Cataract was induced by injection of selenite (19 micromol/kg, s.c.) to 13-day old rats. The lens opacification was monitored by using the slit lamp equipped with an anterior eye segment analysis system (EAS-1000, Nidek). The selenium contents in rat organs were detected fluorimetrically. Liposomes containing DSF (DSF-liposomes) were prepared by the reverse-phase evaporation method. Rat pups were instilled 5 microl of DSF-liposomes into both eyes 4 times per day. Adult rats were administered with DSF suspensions (100 mg/kg) by nasal cannulation. The changes of plasma concentration of diethyldithiocarbamate (DDC), which was a metabolite of disulfiram, were determined by HPLC method. RESULTS: Intraocular treatment with DSF-liposomes prevented the onset of cataract development in rat pups injected with sodium selenite. Treatment with DSF also significantly reduced the selenium contents in plasma at 1 h post-treatment and in the eye at 96 h post-treatment. No significant differences of selenium content in liver and kidney were observed in 13-day old rats instilled with DSF-liposomes or DSF free liposomes. Cataract could not be induced in the 30-days-old rats with the same dose of selenite (19 micromol/kg) and the liver, kidney, and especially eye of the older rats had lower levels of selenium than 13-day old rats. Diethyldithiocarbamate (DDC), an active metabolite of DSF, was decreased in the plasma following a subcutaneous injection of sodium selenite. The selenium concentration in the plasma was decreased by the intranasal administration of DSF suspensions. CONCLUSIONS: Instillation of DSF-liposomes into the eyes of rat pups given a subcutaneous injection of sodium selenite to induce cataracts prevented the formation of cataracts. The mechanism of inhibition may have resulted from a decreased level of selenium in the eyes following the treatment with DSF. On the other hand, as it is known that the cataracts may result from selenite-induced oxidative stress in the lens, DSF and DDC may react as anti-oxidants.

Molecular cloning of the cDNA coding for mouse aldehyde oxidase: tissue distribution and regulation in vivo by testosterone.

The cDNA coding for mouse aldehyde oxidase (AO), a molybdoflavoprotein, has been isolated and characterized. The cDNA is 4347 nt long and consists of an open reading frame predicting a polypeptide of 1333 amino acid residues, with 5' and 3' untranslated regions of 13 and 335 nt respectively. The apparent molecular mass of the translation product in vitro derived from the corresponding cRNA is consistent with that of the monomeric subunit of the AO holoenzyme. The cDNA codes for a catalytically active form of AO, as demonstrated by transient transfection experiments conducted in the HC11 mouse mammary epithelial cell line. The deduced primary structure of the AO protein contains consensus sequences for two distinct 2Fe-2S redox centres and a molybdopterin-binding site. The amino acid sequence of the mouse AO has a high degree of similarity with the human and bovine counterparts, and a significant degree of relatedness to AO proteins of plant origin. Northern blot and in situ hybridization analyses demonstrate that hepatocytes, cardiocytes, lung endothelial or epithelial cells and oesophagus epithelial cells express high levels of AO mRNA. In the various tissues and organs considered, the level of AO mRNA expression is not strictly correlated with the amount of the corresponding protein, suggesting that the synthesis of the AO enzyme is under translational or post-translational control. In addition, we observed sex-related regulation of AO protein synthesis. In the liver of male animals, despite similar amounts of AO mRNA, the levels of the AO enzyme and corresponding polypeptide are significantly higher than those in female animals. Treatment of female mice with testosterone increases the amounts of AO mRNA and of the relative translation product to levels similar to those in male animals.

Purification, cDNA cloning, and tissue distribution of bovine liver aldehyde oxidase.

Aldehyde oxidase was purified to homogeneity from bovine liver and primary structural information obtained by sequencing a series of cleavage peptides permitted the cloning of the corresponding cDNA. The cDNA is 4,630 base pairs long, and it consists of a 102-base pair 5'-untranslated region followed by a 4017-base pair coding region and a 511-base pair 3'-untranslated region. The open reading frame predicts a 1339-amino acid polypeptide with a calculated molecular weight of 147,441, which is consistent with the size of the aldehyde oxidase monomeric subunit. The aldehyde oxidase polypeptide contains consensus sequences for iron-sulfur centers and a molybdopterin binding site. The amino acid sequence deduced from the cDNA shows significant similarity with that of xanthine dehydrogenases from various sources. The primary structure of bovine aldehyde oxidase is remarkably similar (approximately 86%) to that of the translation product of a cDNA recently isolated by Wright et al. (Wright, R. M., Vaitaitis, G. M., Wilson, C. M., Repine, T. B., Terada, L. S., and Repine, J. E. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 10690-10694) and reported to represent human xanthine dehydrogenase. With the help of a monospecific antibody raised against the purified protein and the isolated cDNA, the tissue distribution of the bovine aldehyde oxidase protein and corresponding mRNA was determined. Aldehyde oxidase is expressed at high levels in liver, lung, and spleen, and, at a much lower level, in many other organs.