Characterization of superoxide production from aldehyde oxidase: An important source of oxidants in biological tissues

Abstract

Aldehyde oxidase, a molybdoflavoenzyme that plays an important role in aldehyde biotransformation, requires oxygen as substrate and produces reduced oxygen species. However, little information is available regarding its importance in cellular redox stress. Therefore, studies were undertaken to characterize its superoxide and hydrogen peroxide production. Aldehyde oxidase was purified to >98% purity and exhibited a single band at ∼290 kDa on native polyacrylamide gradient gel electrophoresis. Superoxide generation was measured and quantitated by cytochrome c reduction and EPR spin trapping with p-dimethyl aminocinnamaldehyde as reducing substrate. Prominent superoxide generation was observed with an initial rate of 295 nmol/min/mg. Electrochemical measurements of oxygen consumption and hydrogen peroxide formation yielded values of 650 nmol/min/mg and 355 nmol/min/mg. In view of the ubiquitous distribution of aldehydes in tissues, aldehyde oxidase can be an important basal source of superoxide that would be enhanced in disease settings where cellular aldehyde levels are increased. KeywordsAldehyde oxidase; Xanthine oxidase; Superoxide; Hydrogen peroxide; Electron paramagnetic resonance; Spin trapping; Cytochrome c reduction; Reactive oxygen species; Free radicals; Oxygen consumption Aldehyde oxidase (AO; EC 1.2.3.1) 1 is a prominent member of the molybdenum hydroxylase family of enzymes, which also includes xanthine oxidoreductase (XOR). XOR has two interconvertible forms, xanthine dehydrogenase (XDH; EC 1.1.1.204) and xanthine oxidase (XO; 1. 1.3.22). Both forms of XOR are involved in the catabolism of purines, * To whom correspondence should be addressed: Davis Heart and Lung Research Institute, 473 W. 12th Ave, Room 110G, The Ohio State University, Columbus, OH 43210. Phone: (614) . E-Mail: Jay.Zweier@osumc.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Both XOR and AO are homodimeric consisting of two identical subunits with an approximate molecular mass of 145 kDa each. Each subunit consists of four discrete regions -two N-terminal domains contain distinct [2Fe-2S] centers. A linker peptide connects it to a 40 kDa FAD binding domain that positions the flavin ring in close proximity and a second linker peptide connects the FAD domain with the 85 kDa C-terminal portion of the protein that contains the molybdenum center and the substrate binding pocket [3,4]. The structure of XOR is well conserved among human, chicken, mouse and rat enzymes [5] and the amino acid sequences reveal that the molybdenum binding site is the most conserved region with 94% homo...

The Impact of Single Nucleotide Polymorphisms on Human Aldehyde Oxidase

“…The three bands were analyzed by matrix-assisted laser desorption ionization peptide mapping and identified to be the degradation products of hAOX1. Similar bands also were observed in purified mAOX3 (Mahro et al, 2011) and AOX1 protein preparations of mouse and rat origin (Kundu et al, 2007;Schumann et al, 2009). These products were never detectable upon native PAGE (Kundu et al, 2007;Mahro et al, 2011) and fast protein liquid chromatography.…”

“…Similar bands also were observed in purified mAOX3 (Mahro et al, 2011) and AOX1 protein preparations of mouse and rat origin (Kundu et al, 2007;Schumann et al, 2009). These products were never detectable upon native PAGE (Kundu et al, 2007;Mahro et al, 2011) and fast protein liquid chromatography. All of this suggested that the observed degradation products are generated by the reductive conditions intrinsic to SDS-PAGE.…”

Characterization of the Magnitude and Mechanism of Aldehyde Oxidase-mediated Nitric Oxide Production from Nitrite

“…However, under disease conditions or with alcohol ingestion, these levels are further elevated. We have previously measured that the tissue levels of AO are ϳ60 g/g tissue in liver, 35 g/g in lung, and 5.1 g/g in heart with the activity corresponding to 1.8 units/mg enzyme (21,35). In the presence of physiological levels of NADH (100 M) or aldehyde (50 M), the rate of NO generation followed typical Michaelis-Menten kinetics (Fig.…”

“…DMAC oxidations and absorbance changes were converted to units of enzyme activity (IU) using an extinction coefficient ⑀ ϭ 30.5 mM Ϫ1 cm Ϫ1 . One unit of enzyme activity was defined as the amount of enzyme required to oxidize 1 mol of DMAC/min at 30°C (35). The activity of freshly isolated AO was ϳ1.8 units/mg but declined over time.…”

“…Purification of AO-Rat liver AO was purified as we recently reported (35). AO activity was determined by adding a suitable volume of enzyme solution to 1 ml of 50 mM potassium phosphate buffer, pH 7.8, containing 25 M DMAC as reducing substrate at 30°C and monitoring the decrease in absorbance at 398 nm.…”