Characterization of the Magnitude and Kinetics of Xanthine Oxidase-catalyzed Nitrite Reduction

Abstract

Xanthine oxidase (XO)-catalyzed nitrite reduction with nitric oxide (NO) production has been reported to occur under anaerobic conditions, but questions remain regarding the magnitude, kinetics, and biological importance of this process. To characterize this mechanism and its quantitative importance in biological systems, electron paramagnetic resonance spectroscopy, chemiluminescence NO analyzer, and NO electrode studies were performed. The XO reducing substrates xanthine, NADH, and 2,3-dihydroxybenz-aldehyde triggered nitrite reduction to NO, and the molybdenum-binding XO inhibitor oxypurinol inhibited this NO formation, indicating that nitrite reduction occurs at the molybdenum site. However, at higher xanthine concentrations, partial inhibition was seen, suggesting the formation of a substrate-bound reduced enzyme complex with xanthine blocking the molybdenum site. Studies of the pH dependence of NO formation indicated that XO-mediated nitrite reduction occurred via an acid-catalyzed mechanism. Nitrite and reducing substrate concentrations were important regulators of XO-catalyzed NO generation. The substrate dependence of anaerobic XO-catalyzed nitrite reduction followed Michaelis-Menten kinetics, enabling prediction of the magnitude of NO formation and delineation of the quantitative importance of this process in biological systems. It was determined that under conditions occurring during no-flow ischemia, myocardial XO and nitrite levels are sufficient to generate NO levels comparable to those produced from nitric oxide synthase. Thus, XO-catalyzed nitrite reduction can be an important source of NO generation under ischemic conditions. Xanthine oxidase (XO)-catalyzed nitrite reduction with nitric oxide (NO) production has been reported to occur under anaerobic conditions, but questions remain regarding the magnitude, kinetics, and biological importance of this process. To characterize this mechanism and its quantitative importance in biological systems, electron paramagnetic resonance spectroscopy, chemiluminescence NO analyzer, and NO electrode studies were performed. The XO reducing substrates xanthine, NADH, and 2,3-dihydroxybenz-aldehyde triggered nitrite reduction to NO, and the molybdenum-binding XO inhibitor oxypurinol inhibited this NO formation, indicating that nitrite reduction occurs at the molybdenum site. However, at higher xanthine concentrations, partial inhibition was seen, suggesting the formation of a substrate-bound reduced enzyme complex with xanthine blocking the molybdenum site. Studies of the pH dependence of NO formation indicated that XO-mediated nitrite reduction occurred via an acid-catalyzed mechanism. Nitrite and reducing substrate concentrations were important regulators of XO-catalyzed NO generation. The substrate dependence of anaerobic XO-catalyzed nitrite reduction followed Michaelis-Menten kinetics, enabling prediction of the magnitude of NO formation and delineation of the quantitative importance of this process in biological systems. It was determined that under conditions occurring during no-flow ischemia, myocardial XO and nitrite levels are sufficient to generate NO levels comparable to those produced from nitric oxide synthase. Thus, XO-catalyzed nitrite reduction can be an important source of NO generation under ischemic conditions. nitric-oxide synthase xanthine oxidase xanthine dehydrogenase nicotinamide adenine dinucleotide 2,3-dithydroxybenz-aldehyde nitric oxide N-methyl-d-glucamine dithiocarbamate electron paramagnetic resonance diphenyleneiodonium chloride flavin-adenine dinucleotide phosphate-buffered saline. Nitric oxide is an important regulator of a variety of biological functions, and it also plays important roles in the pathogenesis of cellular injury. It has been generally accepted that NO is solely generated in biological tissues by specific nitric- oxide synthases (NOSs)1 that metabolize arginine to citrulline with the formation of NO. However, previous studies have also demonstrated that NOS-independent generation of NO from nitrite occurs in ischemic tissues, such as the heart, demonstrating that nitrite can be a source rather than a product of NO, particularly under acidic conditions (1Zweier J.L. Wang P. Samouilov A. Kuppusamy P. Nat. Med. 1995; 1: 804-809Crossref PubMed Scopus (679) Google Scholar, 2Zweier J.L. Samouilov A. Kuppusamy P. Biochim. Biophys. Acta. 1999; 1411: 250-262Crossref PubMed Scopus (318) Google Scholar, 3Samouilov A. Kuppusamy P. Zweier J.L. Arch. Biochem. Biophys. 1998; 357: 1-7Crossref PubMed Scopus (140) Google Scholar). Although it is clear that NO formation occurs secondary to nitrite reduction (3Samouilov A. Kuppusamy P. Zweier J.L. Arch. Biochem. Biophys. 1998; 357: 1-7Crossref PubMed Scopus (140) Google Scholar), questions remain regarding the source of the reducing equivalents required and the role of non-NOS enzymes in this process (1Zweier J.L. Wang P. Samouilov A. Kuppusamy P. Nat. Med. 1995; 1: 804-809Crossref PubMed Scopus (679) Google Scholar). Xanthine oxidase (XO) is a ubiquitous enzyme in mammalian cells that plays important roles in both physiological and pathological conditions. It is involved in the catabolism of purine and pyrimidines, oxidizing hypoxanthine to xanthine and xanthine to uric acid. XO also reduces oxygen to superoxide and hydrogen peroxide and is one of the key enzymes responsible for superoxide-mediated cellular injury (4McCord J.M. Roy R.S. Schaffer S.W. Adv. Myocardiol. 1985; 5: 183-189Crossref PubMed Google Scholar). It has a central role in the process of injury that occurs upon reoxygenation of hypoxic cells and tissues (5Allison R.C. Kyle J. Adkins W.K. Prasad V.R. McCord J.M. Taylor A.E. J. Appl. Physiol. 1990; 95: 597-603Crossref Scopus (91) Google Scholar, 6Zweier J.L. Broderick R. Kuppusamy P. Thompson-Gorman S. Lutty G.A. J. Biol. Chem. 1994; 269: 4156-4162Google Scholar, 7Zweier J.L. Kuppusamy P. Lutty G.A. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 4046-4050Crossref PubMed Scopus (561) Google Scholar). XO contains critical flavin, iron sulfur, and molybdenum sites and has some structural similarity with microbial nitrite reductase (8Zhang Z. Naughton D. Winyard P.D. Benjamin N. Biochem. Biophys. Res. Commun. 1998; 249: 767-772Crossref PubMed Scopus (218) Google Scholar). Recently, it has been reported that XO catalyzes reduction of nitrite to NO under hypoxic conditions (8Zhang Z. Naughton D. Winyard P.D. Benjamin N. Biochem. Biophys. Res. Commun. 1998; 249: 767-772Crossref PubMed Scopus (218) Google Scholar, 9Millar T.M. Stevens C.R. Benjamin N. Eisenthal R. Harrison R. Blake D.R. FEBS Lett. 1998; 427: 225-228Crossref PubMed Scopus (413) Google Scholar, 10Godber L.J.B. Doel J.J. Sapkota P.G. Blake R.D. Stevens R.C. Eisenthal R. Harrison R. J. Biol. Chem. 2000; 275: 7757-7763Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar), but a number of important questions have remained regarding the mechanism, substrate specificity, and magnitude of this process. XO exhibits broad specificity, accepting a variety of reducing substrates (11Xia M. Dempski R. Hille R. J. Biol. Chem. 1999; 274: 3323-3330Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). It was first reported that NADH, but not xanthine, can act as an electron donor to XO and catalyze nitrite reduction (8Zhang Z. Naughton D. Winyard P.D. Benjamin N. Biochem. Biophys. Res. Commun. 1998; 249: 767-772Crossref PubMed Scopus (218) Google Scholar, 9Millar T.M. Stevens C.R. Benjamin N. Eisenthal R. Harrison R. Blake D.R. FEBS Lett. 1998; 427: 225-228Crossref PubMed Scopus (413) Google Scholar). Xanthine or hypoxanthine was found to inhibit this NO formation from XO (9Millar T.M. Stevens C.R. Benjamin N. Eisenthal R. Harrison R. Blake D.R. FEBS Lett. 1998; 427: 225-228Crossref PubMed Scopus (413) Google Scholar). However, more recently, it was reported that xanthine can serve as a reducing substrate to stimulate nitrite reduction (10Godber L.J.B. Doel J.J. Sapkota P.G. Blake R.D. Stevens R.C. Eisenthal R. Harrison R. J. Biol. Chem. 2000; 275: 7757-7763Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar). In contrast to this, other investigators reported that XO in the presence of xanthine does not reduce nitrite to NO M. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). In in studies are regarding the of NO formation and of XO for nitrite or the reducing Thus, questions remain regarding the substrate source required as as the of this XO-catalyzed process of NO formation as a of a substrate the important physiological roles of NO in and S. Biochem. PubMed Scopus Google Scholar, J. Full Text PDF PubMed Scopus Google Scholar, J.M. PubMed Scopus Google and the of the of NO and superoxide in biological systems, it is important to the mechanism, magnitude, and role of XO-catalyzed NO generation. To characterize this XO-catalyzed of NO production and its quantitative importance in biological systems, electron paramagnetic resonance spectroscopy, chemiluminescence NO analyzer, and NO electrode studies were performed. The mechanism of NO formation was to occur to nitrite reduction at the molybdenum with or xanthine as reducing The for NADH, and xanthine were enabling prediction of the magnitude of NO formation and delineation of the quantitative importance of this process in biological systems. Xanthine oxidase from oxygen xanthine, diphenyleneiodonium chloride and 2,3-dihydroxybenz-aldehyde were from dithiocarbamate was as PubMed Scopus Google Scholar). was from phosphate-buffered was from were a at were a at with a of of and of NO formation was by the iron complex of and were to the with a in were under anaerobic conditions by in a followed by under to a that was of NO was determined from the of NO of with with NO of (3Samouilov A. Kuppusamy P. Zweier J.L. Arch. Biochem. Biophys. 1998; 357: 1-7Crossref PubMed Scopus (140) Google Scholar). The of the NO production was a nitric oxide a to to In the analyzer, NO is with of and of NO from the were at a of in a with was to the of the to reduce the of during an with was the and NO The of NO was by of the from the and in of the magnitude of NO production was determined from the of the with that from nitrite to (3Samouilov A. Kuppusamy P. Zweier J.L. Arch. Biochem. Biophys. 1998; 357: 1-7Crossref PubMed Scopus (140) Google Scholar). To for of levels of NO formation, a was in the of NO generation by XO were at a with a and NO electrode The the the is to the NO in the The was and with concentrations of NO, NO The electrode was in a with for were at with of the for and of the was to anaerobic conditions the are as of at and the of was by of or was to to the were a NO is paramagnetic and with to the a iron complex with at with a the of the quantitative of NO generation can be Zweier J.L. Biol. Med. 2000; PubMed Scopus Google Scholar, J.L. Wang P. Kuppusamy P. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar, J.M. N. J. Biol. Chem. Full Text PDF PubMed Google Scholar). was to NO generation under anaerobic conditions. In the of of XO and its reducing substrate xanthine to In the of XO but the presence of nitrite a was xanthine and nitrite to a upon of the XO reducing substrates 2,3-dithydroxybenz-aldehyde or xanthine a in the NO was Thus, of the of XO reducing NADH, and xanthine, as electron for XO-catalyzed nitrite reduction and triggered of NO generation under anaerobic conditions. To the of the XO-catalyzed mechanism of NO generation in the of or by the studies were NO generation a specific NO to the of NO generation was from nitrite in the presence of xanthine or However, of XO NO generation was triggered from xanthine or and The magnitude and of NO generation in the presence of xanthine was higher than that with with xanthine and nitrite in the of NO generation was the substrate also NO generation from nitrite in the presence of with magnitude and that from xanthine not Thus, both and NO electrode studies that XO can generate NO production in the presence of substrates that reduce the To that XO-mediated NO generation occurs and to the of this NO studies were a chemiluminescence NO of was at in the NO was from the with and with in the to of the of NO generation as a of (3Samouilov A. Kuppusamy P. Zweier J.L. Arch. Biochem. Biophys. 1998; 357: 1-7Crossref PubMed Scopus (140) Google Scholar). nitrite or nitrite in the presence of xanthine or of NO formation was However, with the of XO in the presence of xanthine or NADH, NO generation was triggered with XO in the presence of xanthine or but in the of nitrite that nitrite was required for NO generation. a higher of XO-mediated NO generation was with xanthine than with was with the electrode the substrate also NO generation from nitrite in the presence of XO with a that with xanthine not The of NO formation from XO-catalyzed nitrite reduction was under anaerobic conditions the NO The dependence of reducing substrate was first determined in the presence of a nitrite of of the reducing xanthine, and NADH, as electron to XO-catalyzed nitrite and of followed Michaelis-Menten reducing the of and were determined by the to the Michaelis-Menten and the for reducing substrate are of reducing the of XO-mediated NO formation was also determined as a of nitrite Michaelis-Menten were as a of nitrite and the are it is to the magnitude of XO-catalyzed NO formation as a of nitrite and reducing substrate and to the quantitative importance of this mechanism of NO generation in a biological substrate levels are The of of XO were to the sites involved in the process of the XO-catalyzed nitrite with reducing to the molybdenum of It was that oxypurinol inhibited XO-catalyzed nitrite reduction of the of reducing substrate inhibition of NO generation was in the presence of xanthine or oxypurinol substrates at the molybdenum of the this that nitrite to the reduced molybdenum site. at the inhibited nitrite reduction was as the reducing and it not inhibit NO generation xanthine was that to and are to reduce the molybdenum that in reduces nitrite to NO. xanthine or are the electron both XO reduction xanthine or and at the molybdenum that oxypurinol inhibit NO It has been reported that xanthine can inhibition of XO to of xanthine to reduced of the enzyme generated in the process of XO-catalyzed oxygen of substrate-bound reduced XO complex electron from the molybdenum to R. R.C. J. Biol. Chem. Full Text PDF PubMed Google Scholar, R. Biochim. Biophys. Acta. PubMed Scopus Google Scholar). In of the questions regarding the role and of xanthine as a reducing substrate for the process of XO-mediated NO formation, NO and studies were to XO-catalyzed NO generation under anaerobic conditions in the presence of concentrations of xanthine Although XO-catalyzed NO generation followed Michaelis-Menten with concentrations of xanthine higher concentrations of xanthine inhibition of NO formation of the in was to be that a XO complex is and the of NO generation from XO-catalyzed nitrite It is that xanthine XO-catalyzed nitrite reduction by to reduced of the in blocking the of nitrite to the molybdenum site. To the magnitude of NO generation that can occur in biological tissues that nitrite concentrations, of NO generation were in the presence of the of concentrations in myocardial (1Zweier J.L. Wang P. Samouilov A. Kuppusamy P. Nat. Med. 1995; 1: 804-809Crossref PubMed Scopus (679) Google Scholar, 2Zweier J.L. Samouilov A. Kuppusamy P. Biochim. Biophys. Acta. 1999; 1411: 250-262Crossref PubMed Scopus (318) Google Scholar, 3Samouilov A. Kuppusamy P. Zweier J.L. Arch. Biochem. Biophys. 1998; 357: 1-7Crossref PubMed Scopus (140) Google Scholar). process of NO generation was that was to that with higher nitrite levels of NO generation of and were for and at xanthine concentrations from to in xanthine inhibition was seen, as was with nitrite ischemic conditions, and pH in tissues, such as the heart, can to levels of or J.L. Samouilov A. Kuppusamy P. Biochim. Biophys. Acta. 1999; 1411: 250-262Crossref PubMed Scopus (318) Google Scholar). In to the NO formation under physiological or pathological conditions and to characterize the mechanism of XO-catalyzed nitrite were to the of pH the magnitude of NO generation. were with of XO in the presence of in it was for of the substrates xanthine, NADH, and that under acidic conditions XO-catalyzed NO generation In under conditions, inhibition was of pH NO generation generation were with XO in the presence of in a were with XO in the presence of In of the critical role of NO in and it is of importance to the of NO formation that occur in biological cells and In to the formation of NO from specific it is clear that nitrite from NO or can be an important source of NO formation, particularly under conditions of and Although NO can be by the process of nitrite that is under acidic conditions, it has been that in ischemic tissues, nitrite is also reduced to NO. have remained regarding the role of non-NOS enzymes in this process of nitrite reduction J.L. Samouilov A. Kuppusamy P. Biochim. Biophys. Acta. 1999; 1411: 250-262Crossref PubMed Scopus (318) Google Scholar). Although it has been reported that XO can reduce nitrite to NO, questions remain regarding the biological importance of this of NO as as the mechanism, magnitude, and substrate of this process. It was first reported that NADH, but not xanthine, can act as an electron donor to XO and catalyze nitrite reduction (8Zhang Z. Naughton D. Winyard P.D. Benjamin N. Biochem. Biophys. Res. Commun. 1998; 249: 767-772Crossref PubMed Scopus (218) Google Scholar, 9Millar T.M. Stevens C.R. Benjamin N. Eisenthal R. Harrison R. Blake D.R. FEBS Lett. 1998; 427: 225-228Crossref PubMed Scopus (413) Google Scholar). Xanthine or hypoxanthine was found to inhibit this NO formation from XO (9Millar T.M. Stevens C.R. Benjamin N. Eisenthal R. Harrison R. Blake D.R. FEBS Lett. 1998; 427: 225-228Crossref PubMed Scopus (413) Google Scholar). However, more recently, it was reported that xanthine can serve as a reducing substrate to stimulate nitrite reduction (10Godber L.J.B. Doel J.J. Sapkota P.G. Blake R.D. Stevens R.C. Eisenthal R. Harrison R. J. Biol. Chem. 2000; 275: 7757-7763Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar). In contrast to this, other investigators reported that XO in the presence of xanthine does not reduce nitrite to NO M. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). In in were regarding the of NO formation and of XO for nitrite or the reducing In of it has not been to the biological and importance of this of NO generation. a of studies chemiluminescence NO analyzer, and NO electrode to the magnitude and of NO formation that to XO-mediated nitrite of that XO does reduce nitrite to NO under anaerobic conditions. It was that of the reducing substrates xanthine, and act as electron to this XO-mediated nitrite reduction The of with the inhibition with that reduced XO was the electron donor to with nitrite and reduction occurring at the molybdenum site. NO generation was inhibited by the NO generation by xanthine or was Thus, xanthine or reduces the molybdenum reduces the flavin, to the questions remain regarding the and for XO-mediated NO generation under anaerobic conditions as a of nitrite and reducing substrate studies reported a of for nitrite reduction in the presence of (9Millar T.M. Stevens C.R. Benjamin N. Eisenthal R. Harrison R. Blake D.R. FEBS Lett. 1998; 427: 225-228Crossref PubMed Scopus (413) Google Scholar). However, a reported of (8Zhang Z. Naughton D. Winyard P.D. Benjamin N. Biochem. Biophys. Res. Commun. 1998; 249: 767-772Crossref PubMed Scopus (218) Google Scholar). more rather than NO generation reported of (10Godber L.J.B. Doel J.J. Sapkota P.G. Blake R.D. Stevens R.C. Eisenthal R. Harrison R. J. Biol. Chem. 2000; 275: 7757-7763Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar). In that the for nitrite was in the presence of with the of (8Zhang Z. Naughton D. Winyard P.D. Benjamin N. Biochem. Biophys. Res. Commun. 1998; 249: 767-772Crossref PubMed Scopus (218) Google Scholar). In contrast with the first studies that enzyme reduction by and the of for nitrite in the presence of xanthine, that the for nitrite is for of the of substrates xanthine and for of the studies to the conditions for NO from the of oxygen the or partial enzyme or in the (10Godber L.J.B. Doel J.J. Sapkota P.G. Blake R.D. Stevens R.C. Eisenthal R. Harrison R. J. Biol. Chem. 2000; 275: 7757-7763Abstract Full Text Full Text PDF PubMed Scopus (334) Google that and to for of the In the of NO the and conditions with of the XO in the presence of NO that is a of the NO generated is for this the that can be and studies were with nitrite concentrations in the of to In in the NO the and this NO and the enabling of the of the enzyme with nitrite concentrations of to in the NO formation the first of XO to the and of the of NO generation by NO electrode Although xanthine was the reducing substrate of XO-catalyzed nitrite xanthine inhibition of NO studies reported that enzyme from of XO to its (10Godber L.J.B. Doel J.J. Sapkota P.G. Blake R.D. Stevens R.C. Eisenthal R. Harrison R. J. Biol. Chem. 2000; 275: 7757-7763Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar). However, this not investigators that XO its as reducing substrate (10Godber L.J.B. Doel J.J. Sapkota P.G. Blake R.D. Stevens R.C. Eisenthal R. Harrison R. J. Biol. Chem. 2000; 275: 7757-7763Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar). also that the presence of or XO-catalyzed NO generation. In a previous that XO in NO from NO in concentrations to Zweier J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). of XO (10Godber L.J.B. Doel J.J. Sapkota P.G. Blake R.D. Stevens R.C. Eisenthal R. Harrison R. J. Biol. Chem. 2000; 275: 7757-7763Abstract Full Text Full Text PDF PubMed Scopus (334) Google be by triggered upon to oxygen at the of that xanthine to inhibit XO by to the molybdenum of the reduced enzyme R. R.C. J. Biol. Chem. Full Text PDF PubMed Google Scholar, R. Biochim. Biophys. Acta. PubMed Scopus Google Scholar), blocking the of nitrite at this enzyme site. has also been demonstrated by (10Godber L.J.B. Doel J.J. Sapkota P.G. Blake R.D. Stevens R.C. Eisenthal R. Harrison R. J. Biol. Chem. 2000; 275: 7757-7763Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar), the to XO-mediated NO generation from nitrite in studies in xanthine were M. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). It has been reported that purine and substrate via a mechanism and that substrate be for (10Godber L.J.B. Doel J.J. Sapkota P.G. Blake R.D. Stevens R.C. Eisenthal R. Harrison R. J. Biol. Chem. 2000; 275: 7757-7763Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar). The of XO reduction by purine and the pH is from to and this of XO reduction to an of nitrite However, that acidic conditions XO-catalyzed nitrite NO generation as the pH was from to or from to suggesting that nitrite reduction via an acid-catalyzed mechanism, to nitrite the pH and it be the substrate of Although the of pH the of XO reduction by reducing it the of XO by the studies it is clear that XO can catalyze the process of NO generation from nitrite under anaerobic or hypoxic conditions to those occurring in ischemic The key questions is the magnitude of this and the levels of NO produced are to have To critical a can be that prediction of the magnitude of XO-catalyzed NO formation and the quantitative importance of this mechanism of NO generation in biological systems. The the in the mechanism. is the is the reduced and is the reduced to the reducing substrate of such as xanthine, and is the It be that for xanthine of nitrite be reduced to NO. The enzyme can be as the of NO generation can be and this can be in the of the Michaelis-Menten are as the of xanthine, more be The enzyme is as The of NO generation can be as are as was that a broad of physiological nitrite concentrations from to nitrite that a to the the of NO generation from XO in the presence of xanthine at higher nitrite levels of a of the was it is that can as a of nitrite The for can be as a and are determined from of the the and Thus, the that remain with a of at nitrite concentrations but at nitrite levels of and with the of (10Godber L.J.B. Doel J.J. Sapkota P.G. Blake R.D. Stevens R.C. Eisenthal R. Harrison R. J. Biol. Chem. 2000; 275: 7757-7763Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar). also that nitrite reduction via an acid-catalyzed that nitrite to reduced of pH the but xanthine and to XO via a mechanism (10Godber L.J.B. Doel J.J. Sapkota P.G. Blake R.D. Stevens R.C. Eisenthal R. Harrison R. J. Biol. Chem. 2000; 275: 7757-7763Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar). NO generation the of of NO XO reduction and nitrite It has been demonstrated that the of XO in the is of Zweier J.L. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar), to of of or of The XO and xanthine dehydrogenase is this In the ischemic heart, xanthine levels from to the of and nitrite levels are (1Zweier J.L. Wang P. Samouilov A. Kuppusamy P. Nat. Med. 1995; 1: 804-809Crossref PubMed Scopus (679) Google Scholar, 2Zweier J.L. Samouilov A. Kuppusamy P. Biochim. Biophys. Acta. 1999; 1411: 250-262Crossref PubMed Scopus (318) Google Scholar, Zweier J.L. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). pH of the of nitrite to is as reported (3Samouilov A. Kuppusamy P. Zweier J.L. Arch. Biochem. Biophys. 1998; 357: 1-7Crossref PubMed Scopus (140) Google Scholar), the of XO-catalyzed nitrite reduction be pH to the of nitrite to (3Samouilov A. Kuppusamy P. Zweier J.L. Arch. Biochem. Biophys. 1998; 357: 1-7Crossref PubMed Scopus (140) Google Scholar), the of XO-catalyzed nitrite reduction be to to catalyzes this process or is to the magnitude of this NO generation be by to an of It has been reported in studies from that nitric oxide synthase NO. xanthine NO generation that of the NO production from conditions with nitrite concentrations, the magnitude of NO production from this be it is also clear that with in xanthine, it be under the acidic and hypoxic conditions occurring during ischemia, does not to NO, this NOS-independent NO generation be of it has been that the occurring during in of to and enzyme A. Zweier J.L. J. Biol. Chem. PubMed Scopus Google Scholar). Thus, XO-catalyzed nitrite reduction be an important source of NO generation in the ischemic NO from nitrite during it serve to via upon it with can in and cellular injury J. Proc. Natl. Acad. Sci. U. S. A. 1990; PubMed Scopus Google Scholar, P. Zweier J.L. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). is the reduced of xanthine and as an electron but oxygen in the of Biochem. J. PubMed Scopus Google Scholar, J. J. Biol. Chem. Full Text PDF PubMed Google Scholar, J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). also contains the as and it can to XO by of to this a in the of the Biochem. J. PubMed Scopus Google Scholar, J. Biol. Chem. Full Text PDF PubMed Google Scholar, J. Biol. Chem. Full Text PDF PubMed Google Scholar). In is higher than the of XO Zweier J.L. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). It was reported that NO generation also occurs from in a to that with XO (10Godber L.J.B. Doel J.J. Sapkota P.G. Blake R.D. Stevens R.C. Eisenthal R. Harrison R. J. Biol. Chem. 2000; 275: 7757-7763Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar). Thus, it is that production of NO from xanthine be higher than that for XO it is clear that XO-mediated NO generation can be an important source of NO under ischemic conditions in biological tissues that levels of the enzyme with nitrite and reducing In tissues such as the and levels of the this be more than for the of the A. N. Google Scholar). the for the the levels of nitrite and enzyme reducing substrates have a critical role in this process. Nitrite is and it is the its is levels of nitrite are at of magnitude this number of that nitrite such as of or in conditions, or this of NO generation. also a reducing such as or Xanthine was the NO generation under anaerobic conditions with a higher than that of Although xanthine concentrations are its is levels of xanthine, in inhibition particularly levels of xanthine this be and this serve a role to of NO. Thus, XO can be an important source of NOS-independent NO generation. anaerobic conditions, XO reduces nitrite to NO at the molybdenum of the enzyme with xanthine, NADH, or substrates to the reducing The for anaerobic nitrite reduction by XO was and it was demonstrated that under conditions of ischemia, the of NO generation is the of nitrite NO production from the enzyme serve as an source of NO under ischemic conditions in NO production from is