Identification of xanthine dehydrogenase/xanthine oxidase as a rat Paneth cell zinc-binding protein

Paneth cells are zinc-containing cells localized in small intestinal crypts, but their function has not been fully elucidated. Previously, we showed that an intravenous injection of diphenylthiocarbazone (dithizone), a zinc chelator, induced selective killing of Paneth cells, and purified a zinc-binding protein in Paneth cells. In the present study, we further characterized one of these proteins, named zinc-binding protein of Paneth cells (ZBPP)-1. Partial amino acid sequences of ZBPP-1 showed identity with rat xanthine dehydrogenase (XD)/xanthine oxidase (XO). Anti-rat XD antibody (Ab) recognized ZBPP-1, and conversely anti ZBPP-1 Ab recognized 85 kDa fragment of rat XD in Western blotting. Messenger RNA and protein levels of XD were consistent with our previous data on the fluctuation of Paneth cell population after dithizone injection. Thus, ZBPP-1 is an 85 kDa fragment of XD/XO in Paneth cells. XD/XO in Paneth cells may play important roles in intestinal function.     

Differences in environment of FAD between NAD-dependent and O2-dependent types of rat liver xanthine dehydrogenase shown by active site probe study

Rat liver deflavoxanthine dehydrogenase has been prepared by incubating native enzyme with calcium chloride. On reconstitution with FAD, about 85% of the original activity is recovered, all which is the O2-dependent type. In contrast, when dithiothreitol-treated deflavoenzyme is incubated with FAD, the recovery of activity is almost the same as above, but most of the recovered activity is of the NAD-dependent type. Deflavoenzyme with or without previous treatment with dithiothreitol was also reconstituted with two artificial FAD analogues, 8-mercapto-FAD and 6-OH-FAD. The difference spectra between the reconstituted enzymes and the initial deflavoenzyme indicate that, in each case, the FAD analogue is bound in its neutral form in dithiothreitol-treated enzyme, whereas it is bound in the anionic form in enzyme without previous dithiothreitol treatment. Furthermore, the protonated forms can be converted into the anionic forms on storage with a concomitant change of activity from the NAD-dependent to the O2-dependent type. This clearly indicates different environments around FAD in the two types of enzyme protein, which are shown to be interconvertible through oxidation-reduction of enzyme cysteinyl residues.     

Comparative study of chicken liver xanthine dehydrogenase and bovine liver xanthine oxidase. dehydrogenase activity of xanthine oxidase (author's transl)]

A method to purify bovine liver xanthine oxidase in described, with which samples of 256-fold specific activity with respect to the initial homogenate are obtained. Bovine liver xanthine oxidase and chicken liver xanthine dehydrogenase with oxygen as electron acceptor exhibit similar profile in pKM and log V versus pH plots. With NAD+ as electron acceptor a different profile in the pKM xanthine plot is obtained for chicken liver xanthine dehydrogenase. However three inflection points at the same pH values appear in all plots. Both enzymes are irreversibly inhibited by pCMB and reversibly by N-ethylmaleimide and by iodoacetamide, with competitive and uncompetitive type inhibitions respectively. These results suggest that NAD+ alters the enzymatic action since its binding to the enzyme antecedes the binding of xanthine to the xanthine oxidase molecule, without undergoing itself any modification. 0.15 M DDT of DTE treatment of bovine liver xanthine oxidase gives to the enzyme a permanent activity with NAD+ without modifying its activity with oxygen. The enzyme thus treated produces parallel straight lines in Lineweaver-Burk plots.     

Role of xanthine oxidase and xanthine dehydrogenase systems in thymocyte apoptosis, induced by papaverine

It has been established that papaverine as well as other xenobiotics (dexamethasone and nitrosodimethylamine) [figure: see text] provoked the thymocyte death like apoptosis. The increase of the quantity of double-strand, single-strand DNA breaks and low molecular weight fragments of DNA preceded cell death. In papaverine-induced process of thymocyte apoptosis the total activity of xanthine oxidase in thymocytes strongly elevated long before their death, the conversion of xanthine dehydrogenase (D-form) to xanthinoxidase (O-form) and accumulation of O-form in the cultural medium took place. Direct stimulating effect of papaverine on O-form of enzyme in thymocyte lysate was revealed. The used digitonin thymocytes were divided into cytoplasmic and structural component fractions. It was shown that about 80% of total xanthinoxidase activity was concentrated in cytoplasma while only 20% of its activity was found in structural components. More higher ratio of xanthinoxidase/xanthindehydrogenase (XO/XDH) was observed and papaverine-induced changes of these enzyme forms activities were expressed more brightly in the structural components, than in the thymocyte cytoplasma. During the process of developing thymocytes apoptosis caused by papaverine the reaction of lipid peroxidation was intensified. XO-hypoxanthin system displaying prooxidant influence on cells increased the cytotoxic effect of papaverine but the presence of allopurinol or catalase and superoxidedismutase decreased it. Besides, cytotoxic action on thymocytes of allopurinol itself as well as hypoxanthin itself was revealed.     

Absence of xanthine oxidase or xanthine dehydrogenase in the rabbit myocardium

We directly measured the activity of the enzymes xanthine oxidase and xanthine dehydrogenase in rabbit and rat hearts, using a sensitive radiochemical assay. Neither xanthine oxidase activity nor xanthine dehydrogenase activity was detected in the rabbit heart. In the rat heart, xanthine oxidase activity was 9.1 +/- 0.5 mIU per gram wet weight and xanthine dehydrogenase activity was 53.0 +/- 1.9 mIU per gram wet weight. These results argue against the involvement of the xanthine oxidase/xanthine dehydrogenase system as a mechanism of tissue injury in the rabbit heart, and suggest that the ability of allopurinol to protect the rabbit heart against hypoxic or ischemic damage must be due to a mechanism other than inhibition of these enzymes.     

Nonlinear kinetics of the xanthine oxidase reaction catalyzed by chicken liver xanthine dehydrogenase

The xanthine oxidase reaction catalyzed by chicken liver xanthine dehydrogenase has been shown to give nonlinear kinetics of the type which has been identified as substrate activation. When a very wide range of substrate (pteridine) concentrations were studied, it was found that a downward deflection in reciprocal plots (substrate activation) occurs in the high region and an upward deflection in the very low region. When product (isoxanthopterin) was included in reaction mixtures, the upward deflection was enhanced and shifted to higher substrate concentration ranges. In addition, reciprocal plots with a second substrate (oxygen) and a product (isoxanthopterin) were nonlinear.     

Phagocyte NADPH-oxidase. Studies with flavin analogues as active site probes in triton X-100-solubilized preparations

NADPH-oxidase of stimulated human neutrophil membranes was solubilized in Triton X-100 and activity reconstituted with FAD, 8-F-FAD, 8-phenyl-S-FAD, and 8-S-FAD. The enzyme had similar affinities for all the flavins with Km values in the 60-80 nM range. Vmax was found to increase 4-fold with increasing redox midpoint potential of the flavin. 8-F-FAD reconstituted with the enzyme was reactive toward thiophenol, suggesting exposure of the 8-position to solvent, a finding supported by unsuccessful attempts to label the enzyme with the photoaffinity probe 8-N3-[32P]FAD. Solubilized oxidase stabilized the red thiolate form of 8-S-FAD, a characteristic of flavoproteins of the dehydrogenase/electron transferase classes which stabilize the blue neutral form of the flavin semiquinone radical.     

Preparation and some properties of 6-substituted flavins as active site probes for flavin enzymes

6-Azidoflavins, 6-thiocyanatoflavins, and 6-mercaptoflavins at the lumiflavin, riboflavin, FMN, and FAD level were prepared from the corresponding 6-aminoflavins and some of their properties investigated. They are bound tightly by apoflavin enzymes which bind either riboflavin, FMN, or FAD. 6-Azidoflavins undergo facile photolysis. One major product was identified as 6-aminoflavin. A further product, which was formed also during acid decomposition of the azide, results from opening of the flavin benzene ring and is proposed to have a lumazine structure. 6-Thiocyanatoflavins are easily converted by dithiothreitol to 6-mercaptoflavins. The latter are stabilized against dimerization in the presence of reducing thiols. 6-Mercaptoflavins have a pK of 5.9, which corresponds to ionization of the 6-SH function. The neutral form is yellow, while the anion is green, due to a long-wavelength band (lambda max approximately 600 nm) extending beyond 700 nm. These properties suggest the use of these 6-substituted flavins for probing the active site of flavin enzymes. Because their reactive substituents are in close proximity to the flavin N(5)-position, these 6-substituted derivatives should also serve as useful probes of the environment around the flavin N(5), a position known to be involved in all flavin-mediated redox processes.     

Synthesis of probes for the active site of leucine dehydrogenase.

A series of novel 3-substituted 2-oxobutanoic acids were prepared and incubated with leucine dehydrogenase giving in one case both a kinetic resolution at C-3 and reductive amination of the ketone. This is the first example of an amino acid dehydrogenase catalysed kinetic resolution and reductive amination.     

Interrelation of xanthine oxidase and dehydrogenase and L-gulonolactone oxidase in animal tissues

There is a correlation between phylogeny and the activities of L-gulonolactone oxidase (LGO), the key enzyme responsible for ascorbic acid (AH2) synthesis in animals and total xanthine oxidase and dehydrogenase [XOD(D/O)], the enzyme responsible for the production of endogenous superoxide radical (O2-.). LGO appears in the kidneys of amphibians and reptiles but livers of mammals. XOD(D/O) also is present mainly in the kidneys of amphibians and reptiles and livers of mammals. AH2 is a potential scavenger of O2-. and it appears that tissue specific expression of LGO takes place to counteract the endogenous O2-. toxicity. The interrelation of XOD(D/O) and LGO was also observed in the liver of rats during prenatal to postnatal development.     

Phosphorylation of xanthine dehydrogenase/oxidase in hypoxia

The enzyme xanthine oxidase (XO) has been implicated in the pathogenesis of several disease processes, such as ischemia-reperfusion injury, because of its ability to generate reactive oxygen species. The expression of XO and its precursor xanthine dehydrogenase (XDH) is regulated at pre- and posttranslational levels by agents such as lipopolysaccharide and hypoxia. Posttranslational modification of the protein, for example through thiol oxidation or proteolysis, has been shown to be important in converting XDH to XO. The possibility of posttranslational modification of XDH/XO through phosphorylation has not been adequately investigated in mammalian cells, and studies have reported conflicting results. The present report demonstrates that XDH/XO is phosphorylated in rat pulmonary microvascular endothelial cells (RPMEC) and that phosphorylation is greatly increased ( approximately 50-fold) in response to acute hypoxia (4 h). XDH/XO phosphorylation appears to be mediated, at least in part, by casein kinase II and p38 kinase as inhibitors of these kinases partially prevent XDH/XO phosphorylation. In addition, the results indicate that p38 kinase, a stress-activated kinase, becomes activated in response to hypoxia (an approximately 4-fold increase after 1 h of exposure of RPMEC to hypoxia) further supporting a role for this kinase in hypoxia-stimulated XDH/XO phosphorylation. Finally, hypoxia-induced XDH/XO phosphorylation is accompanied by a 2-fold increase in XDH/XO activity, which is prevented by inhibitors of phosphorylation. In summary, this study shows that XDH/XO is phosphorylated in hypoxic RPMEC through a mechanism involving p38 kinase and casein kinase II and that phosphorylation is necessary for hypoxia-induced enzymatic activation.     

The role of active site glutamate residues in catalysis of Rhodobacter capsulatus xanthine dehydrogenase

Xanthine dehydrogenase (XDH) from the bacterium Rhodobacter capsulatus catalyzes the hydroxylation of xanthine to uric acid with NAD+ as the electron acceptor. R. capsulatus XDH forms an (alphabeta)2 heterotetramer and is highly homologous to homodimeric eukaryotic xanthine oxidoreductases. Here we first describe reductive titration and steady state kinetics on recombinant wild-type R. capsulatus XDH purified from Escherichia coli, and we then proceed to evaluate the catalytic importance of the active site residues Glu-232 and Glu-730. The steady state and rapid reaction kinetics of an E232A variant exhibited a significant decrease in both kcat and kred as well as increased Km and Kd values as compared with the wild-type protein. No activity was determined for the E730A, E730Q, E730R, and E730D variants in either the steady state or rapid reaction experiments, indicating at least a 10(7) decrease in catalytic effectiveness for this variant. This result is fully consistent with the proposed role of this residue as an active site base that initiates catalysis.     

Distribution of xanthine oxidase and xanthine dehydrogenase specificity types among bacteria.

A diverse collection of xanthine-metabolizing bacteria was examined for xanthine-, 1-methylxanthine-, and 3-methylxanthine-oxidizing activity. Both particulate and soluble fractions of extracts from aerobically grown gram-negative bacteria exhibited oxidation of all three substrates; however, when facultative gram-negative bacteria were grown anaerobically, low particulate and 3-methylxanthine activities were detected. Gram-positive and obligately anaerobic bacteria showed no particulate activity or 3-methylxanthine oxidation. Substrate specificity studies indicate two types of enzyme distributed among the bacteria along taxonomic lines, although other features indicate diversity of the enzyme within these two major groups. The soluble and particulate enzymes from Pseudomonas putida and the enzyme from Arthrobacter S-2 were examined as type examples with a series of purine and analogues differing in the number and position of oxygen groups. Each preparation was active with a variety of compounds, but the compounds and position attacked by each enzyme was different, both from the other enzymes examined and from previously investigated enzymes. The soluble enzyme from Pseudomonas was inhibited in a competitive manner by uric acid, whereas the Arthrobacter enzyme was not. This was correlated with the ability of Pseudomonas, but not Arthrobacter, to incorporate radioactivity from [2-14C]uric acid into cellular material.     

The active site structure of quinohemoprotein amine dehydrogenase inhibited by p-nitrophenylhydrazine

Quinohemoprotein amine dehydrogenase (QH-AmDH) catalyzes the oxidative deamination of aliphatic and aromatic amines. The enzyme from Pseudomonas putida has an alpha beta gamma heterotrimeric structure with two heme c groups in the largest alpha subunit, and a novel quinone cofactor [cysteine tryptophylquinone (CTQ)] and hitherto unknown internal cross-bridges in the smallest gamma subunit. The crystal structure of the enzyme in the complex with the inhibitor [p-nitrophenylhydrazine (pNPH)] has been determined at a 2.0 A resolution.(1) The hydrazone of the cofactor with the inhibitor was nicely modeled into the omit electron density map, identifying the C6 carbonyl group as the reactive site of the cofactor. The Asp33 gamma is unambiguously determined as the catalytic base to abstract the alpha-proton from a substrate, because N beta atom of the inhibitor corresponding to the C alpha atom of the substrate amine is neighbored to Asp33 gamma. The bound inhibitor is completely enclosed in the active site pocket formed by the residues from the beta- and gamma-subunits. The cofactor-inhibitor adduct may be predominantly in the hydrazone with the azo form as a minor component. The binding of the inhibitor causes minor but important conformational changes in the residues surrounding the active site. The inhibitor may have access to the active site pocket through the water-filled crevice between the beta- and gamma-subunits.     

Stimulation of NADH oxidation by xanthine oxidase and polyvanadate in presence of some dehydrogenases and flavin compounds

The rates of NADH oxidation in presence of xanthine oxidase increase to a small and variable extent on addition of high concentrations of lactate dehydrogenase and other dehydrogenases. This heat stable activity is similar to polyvanadate-stimulation with respect to pH profile and SOD sensitivity. Isocitric dehydrogenase (NADP-specific) showed heat labile, SOD-sensitive polyvanadate-stimulated NADH oxidation activity. Polyvanadate-stimulated SOD-sensitive NADH oxidation was also found to occur with riboflavin, FMN and FAD in presence of a non-specific protein, BSA, suggesting that some flavoproteins may possess this activity.     

N-ethylmaleimide inhibits xanthine oxidase activity with no detectable change in xanthine dehydrogenase activity in rabbit liver

The effect of an alkylating agent, N-ethylmaleimide (NEM), on the activities of xanthine oxidase (XO) and xanthine dehydrogenase (XD) in the presence and absence of Cu2+ or trypsin in the cytosolic fraction from rabbit liver was examined. At concentrations ranging from 0.25 to 2.0 microM, allopurinol, which is generally considered to be a XO inhibitor, suppressed the XD activity (41.5-93.4% inhibition) in addition to the XO activity (28.6-88.4% inhibition) under basal conditions, without the addition of Cu2+ or trypsin. In contrast, NEM (100-400 microM) inhibited the XO activity (35.7-85.7% inhibition) without affecting the XD activity. Also, NEM inhibited the Cu2+- and trypsin-induced XO activities, but did not affect the XD activity at the same concentration range. These results demonstrate that NEM can be a selective inhibitor of XO activity in rabbit liver.     

Resonance Raman studies of the flavin and iron-sulfur centers of milk xanthine oxidase

Resonance Raman spectroscopy has been used to study milk xanthine oxidase, an enzyme containing molybdenum, binuclear iron-sulfur clusters, and FAD as cofactors. The contribution of FAD dominates the resonance Raman spectrum at frequencies above 500 cm-1. As expected, no bands assignable to FAD are observed in deflavo xanthine oxidase. The resonance Raman spectrum below 500 cm-1 reveals the contribution of the Fe2S2(Cys)4 groups with frequencies similar to those of adrenodoxin and putidaredoxin. Resonance enhancement profiles of the Fe2S2(Cys)4 clusters indicate intensity variations among the Fe2S2(Cys)4 peaks that are attributed to different excitation wavelength maxima of their bridging and terminal iron-sulfur vibrations. No evidence for Mo-ligand vibrations could be obtained by using excitation wavelengths between 363.8 and 514.5 nm.     

Mechanism of the conversion of xanthine dehydrogenase to xanthine oxidase: identification of the two cysteine disulfide bonds and crystal structure of a non-convertible rat liver xanthine dehydrogenase mutant

Mammalian xanthine dehydrogenase can be converted to xanthine oxidase by modification of cysteine residues or by proteolysis of the enzyme polypeptide chain. Here we present evidence that the Cys(535) and Cys(992) residues of rat liver enzyme are indeed involved in the rapid conversion from the dehydrogenase to the oxidase. The purified mutants C535A and/or C992R were significantly resistant to conversion by incubation with 4,4'-dithiodipyridine, whereas the recombinant wild-type enzyme converted readily to the oxidase type, indicating that these residues are responsible for the rapid conversion. The C535A/C992R mutant, however, converted very slowly during prolonged incubation with 4,4'-dithiodipyridine, and this slow conversion was blocked by the addition of NADH, suggesting that another cysteine couple located near the NAD(+) binding site is responsible for the slower conversion. On the other hand, the C535A/C992R/C1316S and C535A/C992R/C1324S mutants were completely resistant to conversion, even on prolonged incubation with 4,4'-dithiodipyridine, indicating that Cys(1316) and Cys(1324) are responsible for the slow conversion. The crystal structure of the C535A/C992R/C1324S mutant was determined in its demolybdo form, confirming its dehydrogenase conformation.     

NADH oxidase activity of rat liver xanthine dehydrogenase and xanthine oxidase-contribution for damage mechanisms

The involvement of xanthine oxidase (XO) in some reactive oxygen species (ROS) -mediated diseases has been proposed as a result of the generation of O*- and H2O2 during hypoxanthine and xanthine oxidation. In this study, it was shown that purified rat liver XO and xanthine dehydrogenase (XD) catalyse the NADH oxidation, generating O*- and inducing the peroxidation of liposomes, in a NADH and enzyme concentration-dependent manner. Comparatively to equimolar concentrations of xanthine, a higher peroxidation extent is observed in the presence of NADH. In addition, the peroxidation extent induced by XD is higher than that observed with XO. The in vivo-predominant dehydrogenase is, therefore, intrinsically efficient at generating ROS, without requiring the conversion to XO. Our results suggest that, in those pathological conditions where an increase on NADH concentration occurs, the NADH oxidation catalysed by XD may constitute an important pathway for ROS-mediated tissue injuries.