Showing papers on "Xanthine published in 1977"

2,3-Dihydro-2-(guan-7-yl)-3-hydroxy-aflatoxin B1, a Major Acid Hydrolysis Product of Aflatoxin B1-DNA or -Ribosomal RNA Adducts Formed in Hepatic Microsome-mediated Reactions and in Rat Liver in Vivo

Abstract: Summary DNA- and ribosomal RNA-bound aflatoxin B 1 adducts obtained from salmon sperm DNA and rat liver ribosomal RNA with fortified rat and hamster liver microsomes were hydrolyzed with weak acid to yield 2,3-dihydro-2-(guan-7-yl)-3-hydroxy-aflatoxin B 1 as the major product. This product was characterized on the basis of its nuclear magnetic resonance, ultraviolet, and infrared spectra; its hydrolysis to guanine, 2,3-dihydro-2,3-dihydroxy-aflatoxin B 1 , and acid degradation products of the latter; its deamination to a product that yielded xanthine on hydrolysis; and the susceptibility of its nucleic acid precursors to hydrolysis in weak alkali. Acid hydrolysis of the nucleic acid-aflatoxin B 1 , adducts also yielded 2,3-dihydro-2,3-dihydroxy-aflatoxin B 1 and two minor products, the amounts of which were greatly increased if the nucleic acid adducts were previously exposed to weak alkali. One of the latter compounds was tentatively identified as 2,3-dihydro-2-( N 5 -formyl-2,5,6-triamino-4-oxopyrimidin- N 5 -yl)-3-hydroxy-aflatoxin B 1 . Hydrolysis of the hepatic DNA and ribosomal RNA from rats given injections of [ 3 H]aflatoxin B 1 liberated tritiated products that cochromatographed on high-performance liquid chromatography in four solvent systems with the products obtained on hydrolysis of the adducts formed in vitro . The identification of the major product from the adducts formed in vivo with that from the adducts formed in vitro was further shown by the chromatographic identities of the derivatives formed on acetylation, deamination, and acid hydrolysis of the major product from each of the two sources.

Enhancement of platelet function by superoxide anion.

Abstract: During the aerobic conversion of xanthine to uric acid by xanthine oxidase, superoxide anion and hydrogen peroxide are produced along with the hydroxyl radical. Our studies demonstrate that washed human platelets incubated with xanthine and xanthine oxidase aggregated and released [14C]serotonin. Aggregation and release were dependent on the duration of exposure to xanthine oxidase as well as the concentration of enzyme. Both reactions were inhibited by the superoxide scavenger enzyme superoxide dismutase but not by catalase, or the free radical scavenger mannitol, suggesting that they were induced by superoxide anion. Superoxide-dependent release was inhibited by prior incubation of platelets with 1 mM EDTA, 1 micronM prostaglandin E1, or 1 mM dibutyryl cyclic AMP, but was unaffected by 1 mM acetylsalicylic acid or 1 micronM indomethacin. After prolonged incubation with xanthine and xanthine oxidase there was also efflux of up to 15% of intraplatelet 51Cr, a cytosol marker. This leakage was prevented by the addition of catalase to the media but not by superoxide dismutase. Incubation with xanthine and xanthine oxidase did not produce malonyldialdehyde, the three-carbon fatty acid fragment produced during prostaglandin endoperoxide synthesis and lipid peroxidation. Prior exposure of platelets to low fluxes of superoxide anion lowered the threshold for release by subsequent addition of thrombin, suggesting a synergistic effect. We conclude that superoxide-dependent aggregation and release may be a physiologically important method to modulate hemostatic reactions particularly in areas of inflammation or vessel injury which could have high local concentrations of superoxide anion.

Molybdenum cofactors from molybdoenzymes and in vitro reconstitution of nitrogenase and nitrate reductase

Abstract: A molybdenum cofactor (Mo-co) from xanthine oxidase (xanthine:oxygen oxidoreductase, EC 1.2.3.2) can be isolated from the enzyme by a technique that has been used to isolate an iron-molybdenum cofactor (FeMo-co) from component I of nitrogenase. N-Methylformamide is used for the extraction of these molybdenum cofactors. Mo-co from xanthine oxidase activates nitrate reductase (NADPH:nitrate oxidoreductase, EC 1.6.6.2) in an extract from Neurospora crassa mutant strain Nit-1; however, FeMo-co is unable to activate nitrate reductase in strain Nit-1. Mo-co from xanthine oxidase is unable to activate nitrogenase in an extract of Azotobacter vinelandii mutant strain UW45. Inactive component I in this extract can be activated by FeMo-co. These results indicate that nitrate reductase and xanthine oxidase share a common molybdenum cofactor, but this cofactor is different from the molybdenum cofactor in nitrogenase.A. vinelandii synthesizes both Mo-co and FeMo-co. Mo-co is produced when the cells fix N(2) and also when they are repressed for nitrogenase synthesis by growth in a medium containing excess ammonium. However, FeMo-co is not produced when cells are grown in an ammonium-containing medium. Partially purified preparations of component I from A. vinelandii and Klebsiella pneumoniae contain both FeMo-co and Mo-co. The presence of both FeMo-co and Mo-co activities in partially purified preparations of component I explains previous reports of activation of inactive nitrate reductase in strain Nit-1 by acid-treated component I of nitrogenase. The Mo-co can be separated from FeMo-co in these preparations by chromatography on Sephadex G-100 in N-methylformamide. Both FeMo-co and Mo-co are sensitive to oxygen.

The Metabolism of Caffeine by a Pseudomonas putida Strain

Abstract: 1) A bacterium capable of growing aerobically with caffeine (1,3,7-trimethylxanthine) as sole source of carbon and nitrogen was isolated from soil. The morphological and physiological characteristics of the bacterium were examined. The organism was identified as a strain of Pseudomonas putida and is referred to as Pseudomonas putida C1. 15 additional caffeine-degrading bacteria were isolated, and all of them were also identified as Pseudomonas putida strains. The properties of the isolates are discussed in comparison with 6 Pseudomonas putida strains of the American Type Culture Collection. 2) The degradation of caffeine by Pseudomonas putida C1 was investigated; the following 14 metabolites were identified: 3,7-dimethylxanthine (theobromine), 1,7-dimethylxanthine, 7-methylxanthine, xanthine, 3,7-dimethyluric acid, 1,7-dimethyluric acid, 7-methyluric acid, uric acid, allantoin, allantoic acid, ureidoglycolic acid, glyoxylic acid, urea, and formaldehyde. Formaldehyde has been demonstrated to be the product of oxidative N-demethylation mediated by an inducible demethylase. A pathway of caffeine degradation is proposed.

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

Abstract: 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.

Oxidation--reduction potentials of turkey liver xanthine dehydrogenase and the origins of oxidase and dehydrogenase behaviour in molybdenum-containing hydroxylases

Abstract: Redox potentials for the various centres in the enzyme xanthine dehydrogenase (EC 1.2.1.37) from turkey liver determined by potentiometric titration in the presence of mediator dyes, with low-temperature electron-paramagnetic-resonance spectroscopy. Values at 25 degrees C in pyrophosphate buffer, pH 8.2, are: Mo(VI)/Mo(V)(Rapid),-350 +/- 20mV; Mo(V) (Rapid)/Mo(IV), -362 +/- 20mV; Fe-S Iox./Fe-S Ired., -295 +/- 15mV; Fe-S IIox./Fe-S IIred., -292 +/- 15mV; FAD/FADH,-359+-20mV; FADH/FADH2, -366 +/- 20mV. This value of the FADH/FADH2 potential, which is 130mV lower than the corresponding one for milk xanthine oxidase [Cammack, Barber & Bray (1976) Biochem. J. 157, 469-478], accounts for many of the differences between the two enzymes. When allowance is made for some interference by desulpho enzyme, then differences in the enzymes' behaviour in titration with xanthine [Barber, Bray, Lowe & Coughlan (1976) Biochem. J. 153, 297-307] are accounted for by the potentials. Increases in the molybdenum potentials of the enzymes caused by the binding of uric acid are discussed. Though the potential of uric acid/xanthine (-440mV) is favourable for full reduction of the dehydrogenase, nevertheless, during turnover, for kinetic reasons, only FADH and very little FADH2 is produced from it. Since only FADH2 is expected to react with O2, lack of oxidase activity by the dehydrogenase is explained. Reactivity of the two enzymes with NAD+ as electron acceptor is discussed in relation to the potentials.

Determination of Theophylline and Its Metabolites by Liquid Chromatography

Abstract: A high pressure liquid chromatographic assay has been developed for the separation and quantitation of theophylline and its metabolites (1-methyl uric acid, 3-methyl xanthine, and 1,3-dimethyl uric acid), theobromine, and dyphylline in biological fluids, viz. human serum, urine, and saliva. The serum has been rendered protein-free by passage through a filter with a nominal molecular weight limit of 10,000. The filtrate is injected onto a reverse-phase column and the separation achieved by utilizing a polar mobile phase and dyphylline (dihydroxypropyl theophylline) as the internal standard. The results for two normal subjects are included to illustrate the applicability of the technique.

Molybdenum-containing enzymes

Abstract: So little is known about molybdenum enzymes other than milk xanthine oxidase that there is little to be said by way of general conclusions. In all cases where there is direct evidence (except possibly for xanthine dehydrogenase from Micrococcus lactilyticus) it seems that molybdenum in the enzymes does have a redox function in catalysis. For the xanthine oxidases and dehydrogenases and for aldehyde oxidase, the metal is concerned in interaction of the enzymes with reducing substrates. However, for nitrate reductase it is apparently in interaction with the oxidizing substrate that the metal is involved. In nitrogenase the role of molybdenum is still quite uncertain.

Xanthine dehydrogenase from Drosophila melanogaster: a comparison of the kinetic parameters of the pure enzyme from two wild-type isoalleles differing at a putative regulatory site.

Abstract: Xanthine dehydrogenase (XDH) from Drosophila melanogaster has been purified to homogeneity by immunoaffinity chromatography, and its kinetic parameters determined. Drosophila XDH exhibits ordered binding for substrate and NAD+, analogous to the corresponding enzymes from vertebrate sources. The wild-type enzyme exhibits a Km for xanthine of 2.4x10-5 M, and for NAD+ of 4.0x10-5 M. XDH purified from a genetic variant exhibiting elevated levels of enzyme activity has similar kinetic constants. The results provide further evidence that the site of variation in the latter strain results in higher steady state numbers of XDH molecules per fly.

Production of uricase by Candida tropicalis using n-alkane as a substrate.

Abstract: Production of uricase (urate oxidase, EC 1.7.3.3) by n-alkane-utilizing Candida tropicalis pK233 was studied. Although the yeast showed very low enzyme productivity under growing conditions on glucose or an n-alkane mixture (C10 to C13) (less than 2 U/g of dry cells), enzyme formation was enhanced markedly in an induction medium consisting of potassium phosphate buffer, MgSO4, uric acid, and an n-alkane mixture (47 U/g of dry cells) or glucose (21 U/g of dry cells). Of the carbon sources tested, the n-alkane mixture was the most suitable for enzyme production. Appropriate aeration also stimulated uricase formation. In addition to uric acid, xanthine, guanine, adenine, and hypoxanthine were also effective for inducing uricase. Under optimum conditions, the maximum yield of the enzyme was 91 U/g of dry cells. Uricase thus induced was localized in the microbodies of the yeast.

Evidence that superoxide radicals are involved in the hemolytic mechanism of phenylhydrazine.

Abstract: Phenylhydrazine produces in the red blood cell the same effect as the enzymic system xanthine oxidase-xanthine, a superoxide radical generator system. Both effects are inhibited by the enzyme superoxide dismutase.

Purine metabolism in Saccharomyces cerevisiae.

Abstract: The synthesis, interconversion, and catabolism of purine bases, ribonucleosides, and ribonucleotides in wild-type Saccharomyces cerevisiae were studied by measuring the conversion of radioactive adenine, hypoxanthine, guanine, and glycine into acid-soluble purine bases, ribonucleosides, and ribonucleotides, and into nucleic acid adenine and guanine. The pathway(s) by which adenine is converted to inosinate is (are) uncertain. Guanine is extensively deaminated to xanthine. In addition, some guanine is converted to inosinate and adenine nucleotides. Inosinate formed either from hypoxanthine or de novo is readily converted to adenine and guanine nucleotides.

Studies on the intestinal absorption of bovine xanthine oxidase.

Abstract: SummaryCertain aspects of the xanthine oxidase hypothesis of atherosclerosis were studied. Xanthine oxidase activity was determined by liquid scintillation counting of [14C]uric acid formed by the action of the enzyme on [14C]xanthine. Results show that the proposal that xanthine oxidase passes through the stomach and is absorbed intact by the small intestine is implausible. The treatment of milk with a low pH buffer resulted in irreversible loss of xanthine oxidase activity. The incubation of everted rat intestine in buffer and in buffer-milk mixtures showed no difference in enzyme activity in serosal fluid or in intestinal homoge-nates.

Characterization of the E. coli K12 strain AB1157 as impaired in guanine/xanthine metabolism.

Abstract: The widely used E. coli K12 strain AB1157 is impaired in guanine (xnathine) metabolism. Mutants blocked in purine biosynthesis before the stage of inosine monophosphate synthesis do not grow on external guanine or xanthine. The genetic nature of the Gua/Xan lesion is a deletion in the chromosome that covers the proA gene. The lesion causes reduced uptake of guanine.

Elevation of serum xanthine oxidase activity following halothane anesthesia in man.

Abstract: Halothane, but not methoxyflurane, was found to cause specific hepatocellular damage, the hepatotoxicity being prompt but transient. The hepatotoxicity was demonstrated by the elevation in the serum activity of xanthine oxidase, a highly sensitive marker for acute liver damage.

Xanthinuria : study of a large kindred with familial urolithiasis and gout.

Abstract: We have carried out biochemical and clinical studies on a large family in which xanthinuria, xanthine lithiasis, uric acid lithiasis and/or gout were discovered. The analysis of its pedigree has shown that : a) the mode of transmission of xanthinuria is autosomal recessive; b) the occurence of xanthine urolithiasis is likely to be due to the association of a second genetic disorder.

Oxypurine excretion in normal newborn infants.

Abstract: Hypoxanthine, xanthine, uric acid, 6-hydroxypurine, 2,6-hydroxypurine and 2,6,8-hydroxypurine, respectively, are the catabolic products of purine-containing compounds in the human body. Each of the co