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1.
Evidence for the existence of a tyrosyl residue in the nicotinamide adenine dinucleotide binding site of chicken liver xanthine dehydrogenase 总被引:2,自引:0,他引:2
Xanthine-NAD and NADH-methylene blue oxidoreductase activities of chicken liver xanthine dehydrogenase were inactivated by incubation with 5'-[p-(fluorosulfonyl)benzoyl]adenosine (5'-FSBA), an active site directed reagent for nucleotide binding sites. The inactivation reaction displayed pseudo-first-order kinetics. A double-reciprocal plot of inactivation velocity vs. 5'-FSBA concentration showed that 5'-FSBA and enzyme formed a complex prior to inactivation. NAD protected the enzyme from inactivation by 5'-FSBA in a competitive fashion. The modified enzyme had the same xanthine-dichlorophenolindophenol and xanthine-O2 oxidoreductase activities as the native enzyme, and on addition of xanthine to the modified enzyme, bleaching of the spectrum occurred in the visible region. The amount of radioactivity incorporated into the enzyme by incubation with [14C]-5'-FSBA was parallel to the loss of xanthine-NAD oxidoreductase activity, and the stoichiometry was 1 mol/mol of enzyme-bound FAD for complete inactivation. These results indicated that 5'-FSBA modified specifically the binding site for NAD of chicken liver xanthine dehydrogenase. The incorporated radioactivity was released slowly from 14C-labeled enzyme by incubation with dithiothreitol with concomitant restoration of catalytic activity. The modified residue responsible for inactivation was identified as a tyrosine. 相似文献
2.
The components of the active molybdenum cofactor in xanthine oxidase was found. The molybdenum cofactor is responsible for the enzymatic activity of the methyl viologen-nitrate reduction. The inactivation of the methyl viologen-nitrate reductase by cyanide is accompanied by the extraction of sulfur from the enzyme. Cyanide inactivated enzyme can be reactivated by incubation with Na2S. The results suggest that the active site of the methyl viologen-nitrate reductase contains an atom of active sulfur which does not originate from the acid labile sulfur of the Fe/S cluster, neither originate from the organic sulfur of the cysteine residue, nor from the sulfur of persulfide. It is probably another type of inorganic sulfur near the molybdenum atoms, The flavin-free xanthine oxidase may be loss entirely its oxidation activity of xanthine to uric acid. In contrast, the activity of the methyl viologen-nitrate reductase is nearly completly insensitive to the flavinfree treatment. Studies on the Fe-free xanthine oxidase, obtained by metal-binding agent phenanthroline and by acid treatment, revealed Fe (in xanthine oxidase it is the Fe of the Fe/S cluster) is also one of the active conponents, functioning in the methyl viologen-nitrate reductase, besides molybdenum. 相似文献
3.
The effects of xanthine + xanthine oxidase-generated reactive oxygen species (ROS) on rabbit muscle creatine kinase (CK) were studied. Xanthine (0.1 mM) + xanthine oxidase (30 mU/ml) inhibited activity of rabbit muscle CK (1.2mU/ml). Catalase (100/ml), but not SOD (100 U/ml), deferoxamine (100μM) or mannitol (20 mM), protected CK from inactivation; suggesting that H2O2 was responsible for inactivation. These results were different from previously reported findings on bovine heart CK that superoxide radicals inactivate the enzyme. Thus, enzymes with homologous structures may have different reactivities to different ROS. H2O2-induced inactivation of rabbit muscle CK was accompanied by a decrease in its thiol group content, whereas no significant changes in the protein structure were detected by SDS-PAGE or carbonyl content. These results suggest that oxidation of -SH groups by H2O2 seems to be a major mechanism of activation of rabbit muscle CK by xanthine + xanthine oxidase. Such inactivation of CK by H2O2 may be important in ROS-induced pathology. 相似文献
4.
《Free radical research》2013,47(2):131-136
The effects of xanthine + xanthine oxidase-generated reactive oxygen species (ROS) on rabbit muscle creatine kinase (CK) were studied. Xanthine (0.1 mM) + xanthine oxidase (30 mU/ml) inhibited activity of rabbit muscle CK (1.2mU/ml). Catalase (100/ml), but not SOD (100 U/ml), deferoxamine (100μM) or mannitol (20 mM), protected CK from inactivation; suggesting that H2O2 was responsible for inactivation. These results were different from previously reported findings on bovine heart CK that superoxide radicals inactivate the enzyme. Thus, enzymes with homologous structures may have different reactivities to different ROS. H2O2-induced inactivation of rabbit muscle CK was accompanied by a decrease in its thiol group content, whereas no significant changes in the protein structure were detected by SDS-PAGE or carbonyl content. These results suggest that oxidation of -SH groups by H2O2 seems to be a major mechanism of activation of rabbit muscle CK by xanthine + xanthine oxidase. Such inactivation of CK by H2O2 may be important in ROS-induced pathology. 相似文献
5.
Purification and substrate inactivation of xanthine dehydrogenase from Chlamydomonas reinhardtii. 总被引:1,自引:0,他引:1
R Pérez-Vicente J M Alamillo J Cárdenas M Pineda 《Biochimica et biophysica acta》1992,1117(2):159-166
Xanthine dehydrogenase (XDH) from the unicellular green alga Chlamydomonas reinhardtii has been purified to electrophoretic homogeneity by a procedure which includes several conventional steps (gel filtration, anion exchange chromatography and preparative gel electrophoresis). The purified protein exhibited a specific activity of 5.7 units/mg protein (turnover number = 1.9 .10(3) min-1) and a remarkable instability at room temperature. Spectral properties were identical to those reported for other xanthine-oxidizing enzymes with absorption maxima in the 420-450 nm region and a shoulder at 556 nm characteristic of molybdoflavoproteins containing iron-sulfur centers. Chlamydomonas XDH was irreversibly inactivated upon incubation of enzyme with its physiological electron donors xanthine and hypoxanthine, in the absence of NAD+, its physiological electron acceptor. As deduced from spectral changes in the 400-500 nm region, xanthine addition provoked enzyme reduction which was followed by inactivation. This irreversible inactivation also took place either under anaerobic conditions or whenever oxygen or any of its derivatives were excluded. Adenine, 8-azaxanthine and acetaldehyde which could act as reducing substrates of XDH were also able to inactivate it upon incubation. The same inactivating effect was observed with NADH and NADPH, electron donors for the diaphorase activity associated with xanthine dehydrogenase. In addition, partial activities of XDH were differently affected by xanthine incubation. We conclude that xanthine dehydrogenase inactivation by substrate is due to an irreversible process affecting mainly molybdenum center and that sequential and uninterrupted electron flow from xanthine to NAD+ is essential to maintain the enzyme in its active form. 相似文献
6.
Inactivation of glutathione peroxidase by superoxide radical 总被引:28,自引:0,他引:28
The selenium-containing glutathione peroxidase, when in its active reduced form, was inactivated during exposure to the xanthine oxidase reaction. Superoxide dismutase completely prevented this inactivation, whereas catalase, hydroxyl radical scavengers, or chelators did not, indicating that O2 was the responsible agent. Conversion of GSH peroxidase to its oxidized form, by exposure to hydroperoxides, rendered it insensitive toward O2. The oxidized enzyme regained susceptibility toward inactivation by O2 when reduced with GSH. The inactivation by O2 could be reversed by GSH; however, sequential exposure to O2 and then hydroperoxides caused irreversible inactivation. Reactivity toward CN- has been used as a measure of the oxidized form of GSH peroxidase, whereas reactivity toward iodoacetate has been taken as an indicator of the reduced form. By these criteria both O2 and hydroperoxides convert the reduced form to oxidized forms. A mechanism involving oxidation of the selenocysteine residue at the active site has been proposed to account for these observations. 相似文献
7.
L S Terada J A Leff D M Guidot I R Willingham J E Repine 《Free radical biology & medicine》1991,10(1):61-68
The mechanism of xanthine oxidase (XO) inactivation by hydrogen peroxide (H2O2) and its biologic significance are unclear. We found that addition of increasing concentrations of H2O2 progressively decreased xanthine oxidase activity in the presence but not the absence of xanthine in vitro. Inactivation of XO by H2O2 was also enhanced by anaerobic reduction of XO by xanthine. Inactivation of XO by H2O2 was accompanied by production of hydroxyl radical (.OH), measured as formation of formaldehyde from dimethylsulfoxide (DMSO). In contrast, addition of H2O2 to deflavo XO did not produce .OH. Inactivation of XO by H2O2 was decreased by simultaneous addition of the .OH scavenger, DMSO. However, inactivation of XO by H2O2 and formation of .OH were not decreased following addition of the metal chelator. DETAPAC, and/or the O2 scavenger, superoxide dismutase. The results suggest that inactivation of XO by H2O2 occurs by production of .OH following direct reduction of H2O2 by XO at the flavin site. 相似文献
8.
Human hypoxanthine guanine phosphoribosyltransferase (HGPRT) lacks the ability to phosphoribosylate xanthine, a property exhibited by HGPRTs from many parasitic protozoa. Using random mutagenesis we have obtained a mutant, F36L, of human HGPRT that phosphoribosylates xanthine. Examination of the structure indicates that F36 does not make direct contact with the purine, but long-range modulation via loop IV, a segment contacting purine at C2 position, could influence substrate specificity. Expanded substrate specificity to include xanthine probably arises from increased flexibility of loop IV as a consequence of mutation at F36. Mutation of the corresponding residue, L44 in Plasmodium falciparum HGPRT, also results in alteration of K(m) and k(cat) for xanthine, substantiating its role in affecting purine base affinity. Our studies show that mutation of this residue in the core of the protein also affects the stability of both enzymes. 相似文献
9.
Papakostas K Georgopoulou E Frillingos S 《The Journal of biological chemistry》2008,283(20):13666-13678
Transmembrane helix XII of UapA, the major fungal homolog of the nucleobase-ascorbate transporter (NAT/NCS2) family, has been proposed to contain an aromatic residue acting as a purine-selectivity filter, distinct from the binding site. To analyze the role of helix XII more systematically, we employed Cys-scanning mutagenesis of the Escherichia coli xanthine-specific homolog YgfO. Using a functional mutant devoid of Cys residues (C-less), each amino acid residue in sequence 419ILPASIYVLVENPICAGGLTAILLNIILPGGY450 (the putative helix XII is underlined) was replaced individually with Cys. Of the 32 single-Cys mutants, 25 accumulate xanthine to 80-130% of the steady state observed with C-less YgfO, six (P421C, S423C, I424C, Y425C, L427C, G436C) accumulate to low levels (15-40%), and I432C is inactive. Immunoblot analysis shows that P421C and I432C display low expression in the membrane. Extensive mutagenesis reveals that replacement of Ile-432 with equally or more bulky side chains abolishes active transport without affecting expression, whereas replacement with smaller side chains allows activity but impairs affinity for the analogues 1-methyl and 6-thioxanthine. Only three of the single-Cys mutants of helix XII (V426C, N430C, and N443C) are sensitive to inactivation by N-ethylmaleimide. N430C is highly sensitive, with an IC50 of 10 microm, and is completely protected against inactivation in the presence of 2-thioxanthine, a high affinity substrate analogue. Other xanthine analogues are poorly bound by N430C, whereas replacement of Asn-430 with Thr inactivates the permease. The findings suggest that Ile-432 and Asn-430 of helix XII are crucial for purine uptake and affinity, and Asn-430 is probably at the vicinity of the binding site. 相似文献
10.
Studies have been made on the possible involvement of malondialdehyde (MDA) and (E)-4-hydroxynon-2-enal (HNE), two terminal compounds of lipid peroxidation, in modifying xanthine oxidoreductase activity through interaction with the oxidase (XO) and/or dehydrogenase (XDH) forms. The effect of the two aldehydes on XO (reversible, XO(rev), and irreversible, XO(irr)) and XDH was studied using xanthine oxidase from milk and xanthine oxidoreductase partially purified from rat liver. The incubation of milk xanthine oxidase with these aldehydes resulted in the inactivation of the enzyme following pseudo-first-order kinetics: enzyme activity was completely abolished by MDA (0.5-4 mM), while residual activity (5% of the starting value) associated with an XO(irr) form was always observed when the enzyme was incubated in the presence of HNE (0.5-4 mM). The addition of glutathione to the incubation mixtures prevented enzyme inactivation by HNE. The study on the xanthine oxidoreductase partially purified from rat liver showed that MDA decreases the total enzyme activity, acting only with the XO forms. On the contrary HNE leaves the same level of total activity but causes the conversion of XDH into an XO(irr) form. 相似文献
11.
Inhibition of xanthine oxidase by various aldehydes 总被引:1,自引:0,他引:1
The inactivation of bovine milk xanthine oxidase by various aldehydes has been investigated. For each aldehyde, the inactivation reaction gives rise to a unique molybdenum(V) electron paramagnetic resonance signal from xanthine oxidase (the Inhibited signal). Of the aldehydes tested, only a few (mainly aromatic) failed to undergo this reaction. The g values of the Inhibited signals vary systematically from one aldehyde to another. As the substituents of the alpha-carbon atom become more electron withdrawing, so the gav increases. The inactivation rate depends on both enzyme and aldehyde concentration. Oxygen or another oxidizing substrate is also required for inhibition by 3-pyridinecarboxaldehyde and butyraldehyde but not formaldehyde. Reactivation of xanthine oxidase inhibited by an aldehyde occurs spontaneously after removal of excess aldehyde. For butyraldehyde or 3-pyridinecarboxaldehyde, greater than 95% recovery of activity was observed. The rate of reactivation is dependent both on the nature of the molecule bearing the aldehyde group and on a pK (6.6) of the complex with the enzyme. Evidence is presented that the modifying aldehyde in the Inhibited signal-giving species has (contrary to earlier assumptions) not been oxidized. These results are discussed in relation to the structure of the molybdenum center, and a mechanism for the inhibiting reaction is suggested. 相似文献
12.
Phosphate was reported to be an inhibitor of copper- and zinc-containing superoxide dismutase (SOD) [de Freitas, D.M., & Valentine, J.S. (1984) Biochemistry 23, 2079-2082]. Thus SOD activity, in 50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (pH 7.4), was decreased by approximately 50% when the assay was made 10 mM in phosphate, and the ionic strength was adjusted with sodium fluoride. The inhibitory effect of phosphate was attributed to the neutralization of the positive charge on the guanidino residue of Arg-141. We have reexamined the effects of phosphate inhibition of SOD and found that the enzyme has identical activity in phosphate or HEPES buffer when the ionic strength is adjusted with NaBr. The putative inhibitory effect of phosphate appears to have been due to fluoride inhibition of the superoxide generating system of xanthine/xanthine oxidase. We have confirmed this result by using a photochemical generation of O2- in addition to the enzymatic generation of O2-. Chemical modification of the lysine residues to homoarginines does not affect the activity of the enzyme and does not impart a phosphate sensitivity. Chemical modification with phenylglyoxal caused approximately 80% inactivation of the native enzyme and 90% inactivation of the O-methylisourea-modified enzyme. Our results suggest that phosphate does not inhibit the copper- and zinc-containing superoxide dismutase (Cu,Zn-SOD) beyond the expectations of its effect on ionic strength. 相似文献
13.
1. The presence of xanthine was required for the inhibition of bovine milk xanthine oxidase by o-iodosobenzoate, iodoacetamide, hydrogen peroxide or p-chloromercuribenzoate. 2. Inactivation by p-chloromercuribenzoate was very rapid, was reversed by cysteine and was less in the presence of FAD. Lineweaver-Burk plots showed that the inactivation by p-chloromercuribenzoate was competitive with substrate. 3. Inactivation by o-iodosobenzoate, iodoacetamide or hydrogen peroxide could not be reversed by cysteine or xanthine. However, the presence of xanthine during the incubation with inhibitor protected the enzyme against o-iodosobenzoate but not against iodoacetamide or hydrogen peroxide. 4. p-Chloromercuribenzoate protected the enzyme against inactivation by hydrogen peroxide. 相似文献
14.
Glucose-6-phosphate dehydrogenase purified from Saccharomyces cerevisiae is rapidly inactivated by diethylpyrocarbonate at pH 6.8 and 30 degrees C with a concomitant increase in absorbance at 242 nm. The second-order rate constant for inactivation was calculated to be 487.8 M-1 min-1. The pH dependence of inactivation suggests the involvement of an amino acid residue having a pKa of 6.77. These results indicate that the inactivation is due to the modification of a histidine residue(s). In the presence of substrate, glucose-6-phosphate or NADP+, the rate of inactivation is decreased, indicating that the essential histidine residue(s) is located at the active site, possibly at the region of overlap of substrates at the binding site. 相似文献
15.
Localization of xanthine oxidase in mammary-gland epithelium and capillary endothelium 总被引:22,自引:0,他引:22
Xanthine oxidase, an iron-sulfur molybdenum flavoprotein known to generate superoxide radical, was demonstrated in several bovine tissues. The enzyme (155 kd polypeptide) was purified from bovine milk lipid globules and antibodies were raised that allowed precipitation of the enzyme without inactivation of enzymatic activity. By immunolocalization techniques at light and electron microscope levels, the antigen was found in milk-secreting epithelial cells but not in epithelial cells of several other tissues. In a number of tissues, including mammary gland, liver, heart, lung and intestine, antibodies to xanthine oxidase stained only endothelial cells of capillaries, including sinusoids, but not endothelia of larger blood vessels and endocard. In both milk-secreting epithelial and capillary endothelial cells, xanthine oxidase was distributed throughout the cytoplasm. Results from biochemical and immunological studies suggest that xanthine oxidase is similar in the various tissues examined and may serve similar redox functions. 相似文献
16.
1. Reduction of chicken liver xanthine dehydrogenase (xanthine: NAD+ oxidoreductase, EC 1.2.1.37) by xanthine under anaerobic condition proceeded in two phases. This biphasicity may be due to functional and non-functional enzymes in the enzyme preparation. 2. Cyanolysis of a persulfide group of chicken liver enzyme resulted in an inactivation of the enzyme. The non-functional enzyme in the standard enzyme preparation was found to lack persulfide groups at the active sites. 3. The remaining NADH-Methylene Blue oxidoreductase activity, after KI treatment of the xanthine-reduced enzyme of a high flavin activity ratio, is not at the level of 50% of the initial activity, differing from the report suggesting non-equivalence of FAD chromophores. 4. The findings in the present report indicate that FAD chromophores of chicken liver enzyme are essentially equivalent. 相似文献
17.
Hydrogen peroxide damages the zinc-binding site of zinc-deficient Cu,Zn superoxide dismutase 总被引:4,自引:0,他引:4
Mutations in Cu,Zn superoxide dismutase (Cu,Zn SOD) account for approximately 20% of cases of familial amyotrophic lateral sclerosis (ALS), a late-onset neurodegenerative disease affecting motor neurons. These mutations decrease protein stability and lower zinc affinity. Zinc-deficient SOD (Cu,E SOD) has altered redox activities and is toxic to motor neurons in vitro. Using bovine SOD, we studied the effects of hydrogen peroxide (H(2)O(2)) on Cu,E SOD and Cu,Zn SOD. Hydrogen peroxide treatment of Cu,E SOD inactivated zinc binding activity six times faster than superoxide dismutase activity, whereas inactivation of dismutase activity occurred at the same rate for both Cu,Zn SOD and Cu,E SOD. Zinc binding by Cu,E SOD was also damaged by simultaneous generation of superoxide and hydrogen peroxide by xanthine oxidase plus xanthine. Although urate, xanthine, and ascorbate can protect superoxide dismutase activity of Cu,Zn SOD from inactivation, they were not effective at protecting Cu,E SOD. Hydrogen peroxide induced subtle changes in the tertiary structure but not the secondary structure of Cu,E SOD as detected by near and far UV circular dichroism. Our results suggest that low levels of hydrogen peroxide could potentially enhance the toxicity of zinc deficient SOD to motor neurons in ALS by rendering zinc loss from SOD irreversible. 相似文献
18.
Xanthine oxidase employs four electron transport sites (flavin adenine dinucleotide (FAD), molybdenum, and two FeS centers) in catalyzing a variety of redox reactions. To determine whether the redox sites reside in independent domains of the enzyme, the temperature of heat inactivation of each site's catalytic activity was determined, except that no attempt was made to distinguish between the two FeS sites. In the oxidase form of xanthine oxidase, the order of thermal stabilities was Mo greater than FAD greater than FeS, while after conversion to its dehydrogenase form the above ranking was Mo greater than FeS greater than FAD. The small but reproducible difference in heat inactivation temperatures among the redox sites demonstrated that the sites are located in separate domains of the enzyme. To confirm the above segregation of redox centers, the temperature of heat-induced release of each redox cofactor from its site on the enzyme was examined. These temperatures were found to be different for each redox cofactor and agreed closely with the heat inactivation temperatures measured above. The data thus demonstrate that both heat inactivation and cofactor release derive from thermal unfolding of independent domains. Using a technique termed "thermal digestion analysis," the FAD domain was located in a approximately equal to 42,000-Da tryptic fragment, while the FeS and Mo domains were isolated in a trypsin-resistant 92,000-Da fragment. We conclude that xanthine oxidase is constructed in modular fashion with the redox sites located in independent structural domains. 相似文献
19.
An auxiliary beta2 subunit, when coexpressed with Slo alpha subunits, produces inactivation of the resulting large-conductance, Ca(2+) and voltage-dependent K(+) (BK-type) channels. Inactivation is mediated by the cytosolic NH(2) terminus of the beta2 subunit. To understand the structural requirements for inactivation, we have done a mutational analysis of the role of the NH(2) terminus in the inactivation process. The beta2 NH(2) terminus contains 46 residues thought to be cytosolic to the first transmembrane segment (TM1). Here, we address two issues. First, we define the key segment of residues that mediates inactivation. Second, we examine the role of the linker between the inactivation segment and TM1. The results show that the critical determinant for inactivation is an initial segment of three amino acids (residues 2-4: FIW) after the initiation methionine. Deletions that scan positions from residue 5 through residue 36 alter inactivation, but do not abolish it. In contrast, deletion of FIW or combinations of point mutations within the FIW triplet abolish inactivation. Mutational analysis of the three initial residues argues that inactivation does not result from a well-defined structure formed by this epitope. Inactivation may be better explained by linear entry of the NH(2)-terminal peptide segment into the permeation pathway with residue hydrophobicity and size influencing the onset and recovery from inactivation. Examination of the ability of artificial, polymeric linkers to support inactivation suggests that a variety of amino acid sequences can serve as adequate linkers as long as they contain a minimum of 12 residues between the first transmembrane segment and the FIW triplet. Thus, neither a specific distribution of charge on the linker nor a specific structure in the linker is required to support the inactivation process. 相似文献
20.
Inhibition of xanthine oxidase by folic acid was reexamined after complete removal of the contaminant which was responsible for time-dependent inactivation (Lewis, A. S., Murphy, L., Mcalla, C., Fleary, M., and Purcell, S. (1984) J. Biol. Chem. 259, 12-15; Spector, T., and Ferone, R. (1984) J. Biol. Chem. 259, 10784-10786). From turnover experiments using stopped flow equipment with a limited amount of xanthine and excess oxygen, and from kinetic analyses with an oxygen electrode, folic acid was found to be an inhibitor of xanthine oxidase. The inhibition was competitive with xanthine with a Ki value of 4.2 X 10(-5) M. From the behavior of the enzyme in affinity chromatography using a Sepharose 4B/folate column, folic acid was also confirmed to be a competitive inhibitor of xanthine oxidase. When enzyme which had been pretreated with oxipurinol was applied to the affinity column, two fractions of xanthine oxidase were separated. The first fraction was found to contain the fully active form (double-active dimers) from the analyses of spectral changes on addition of xanthine, oxipurinol titration, and ESR slow signal, whereas the second fraction was assumed to contain mixed dimers and double-inactive dimers. The ratio of the content of the first fraction to that of the second fraction supports the hypothesis that there are three enzyme species and that there is no interaction either in catalytic activity or in sulfuration or desulfuration reactions between the two subunits. 相似文献