Conversion of xanthine dehydrogenase into oxidase and its role in reperfusion injury

Abstract

Xanthine dehydrogenases from various sources are proteins of molecular mass 300 kDa and are composed of two identical independent subunits; each subunit contains one molybdopterin, two non-identical 2Fe/2S centres and FAD [ 1,2]. T h e mammalian enzyme exists originally as xanthine dehydrogenase, but is converted into xanthine oxidase during extraction or purification procedures [3]. Xanthine dehydrogenase is characterized by high xanthine/NAD+ activity and low xanthine/Oz activity, whereas xanthine oxidase is characterized by high xanthine/OZ activity and negligible xanthine/NAD+ activity. This conversion occurs either reversibly by thiol oxidation of the protein molecule or irreversibly by proteolytic cleavage [4]. In contrast with the mammalian enzyme, xanthine dehydrogenase from chicken liver is not converted into xanthine oxidase by either thiol oxidant or proteinase treatment. T h e full amino acid sequences of the enzymes from human liver [5], rat liver [6], mouse liver [7], bovine milk [8], chicken liver [9] and Drosophifu [ 101 have been determined by cDNA cloning. All known xanthine-oxidizing enzymes consist of approx. 1330 residues, except for the chicken enzyme which consists of 1358 residues because of insertion of 23 amino acid residues in the N-terminal region. Although the amino acid sequences are highly homologous among the rat, mouse, bovine and human enzymes, with about 90% identity [5-81, weaker homology was observed between the mammalian enzyme and the Drosophila enzyme, with 52% identity [6,10], and between the chicken liver and mammalian enzymes, with 70% identity [9]. By limited proteolysis of the rat enzyme with trypsin, the enzyme is converted into a xanthine oxi-dase type with concomitant cleavage into three fragments (20, 40 and 85 kDa) [6]. These fragments are only dissociated under denaturating conditions such as in the presence of high concentrations of guanidinium chloride [6], suggesting that the three fragments associate closely with each other. Determination of the N-terminal amino acid sequences of each fragment led to the assignment of the 20 kDa fragment to the N-terminal portion, the 85 kDa fragment to the C-terminal portion and the 40 kDa fragment to the intermediate portion. The chicken enzyme was also cleaved into three fragments in a similar way, but the enzyme is not converted into the oxidase [9]. Comparative alignment of the deduced protein sequences has been attempted in order to identify the possible locations of the redox active centres. Sequence comparison [5-91 and chemical-modification studies suggested that each redox centre is located in different domains, i.e. the two iron-sulphur centres are in the 20 kDa domain, the FAD is in the 40 kDa domain and the molybdopterin is in the 85 kDa domain [6,9]. These conclusions are consistent with the results obtained from X-ray-crystallographic analysis of aldehyde oxidoreductase from Desulfovibrio g&as which is another molybdenum-containing iron-sulphur protein [ 1 11. There is high sequence homology between xanthine ox...

NADH oxidase activity of rat and human liver xanthine oxidoreductase: potential role in superoxide production

“…Most interest was mainly focused on freeradical metabolism, mediated through hypoxanthine/xanthine oxidation by XO, in biochemical mechanisms associated with a wide variety of human diseases. This ''new'' ROS-generating pathway, of XOR and NADH, must then be reevaluated in those clinical conditions where an increase in NADH is expected, such as in diseases where hypoxic and reperfusion cycles exist, in ethanol hepatotoxicity and in diabetes [12,14,[51][52][53][54].…”

“…Normal processes of metabolism continuously form ROS, and many of them may have useful physiological functions, such as in host defence against invading pathogens, and as mediators of signal transduction [10,11]. Their overproduction, however, can play a major role in several pathological conditions [12][13][14][15][16]. Moreover, it was recently shown that XOR can catalyse the reduction of nitrates and nitrites, giving rise to both • NO [17,18] and peroxynitrite (ONOO -) [19].…”

Therapeutic Effects of Xanthine Oxidase Inhibitors: Renaissance Half a Century after the Discovery of Allopurinol

“…Inherited XOR deficiency leads to xanthinuria and a characteristic multiple organ failure syndrome characterized by the deposition of xanthine in various tissues. The detailed biochemistry of XO and XDH conversion of XDH to XO has been subject to several reviews and research papers (Nishino, 1994;Nishino et al, 1997Nishino et al, , 2005Pritsos, 2000;Borges et al, 2002;Ilich and Hille, 2002;McManaman and Bain, 2002;Meneshian and Bulkley, 2002). It is interesting to note that recent work has shown that XO is regulated on the transcriptional and post-transcriptional levels (Hoidal et al, 1997;Terada et al, 1997;Hassoun et al, 1998;Page et al, 1998;Xu et al, 2000;Ghio et al, 2002).…”

Inhibition of Xanthine Oxidase and Xanthine Dehydrogenase by Nitric Oxide

“…generation, however, was suggested to increase when the apparent ratio of XDH to XO changes. That is, as XDH is inhibited by the accumulated NADH during ischemia, accumulated hypoxanthine is utilized more by XO than would be the case under normal conditions, and increased superoxide production may occur upon reperfusion even though conversion from XDH to XO does not take place (41,42 …”