Showing papers on "Xanthine published in 1981"

Selection for animal cells that express the Escherichia coli gene coding for xanthine-guanine phosphoribosyltransferase

Abstract: Cultured monkey (TC7) and mouse (3T6) cells synthesize an Excherichia coli enzyme, xanthine-guanine phosphoribosyltransferase (XGPRT; 5-phospho-alpha-D-ribose-1-diphosphate:xanthine phosphoribosyltransferase, EC 2.4.2.22), after transfection with DNA vectors carrying the corresponding bacterial gene, Ecogpt. In contrast to mammalian cells, which do not efficiently use xanthine for purine nucleotide synthesis, cells that produce E. coli XGPRT can synthesize GMP from xanthine via XMP. After transfection with vector-Ecogpt DNAs, surviving cells producing XGPRT can be selectively grown with xanthine as the sole precursor for guanine nucleotide formation in a medium containing inhibitors (aminopterin and mycophenolic acid) that block de novo purine nucleotide synthesis. Cells transformed for Ecogpt arise with a frequency of 10(-4) to 10(-5); they appear to be genetically stable in as much as there is no discernible decrease in XGPRT formation or loss on their ability to grow in selective medium after propagation in nonselective medium. Although several of the vector-gpt DNAs can replicate in monkey and mouse cells, none of the transformants contain autonomously replicating vector-gpt DNA. Rather, the gpt transformants contain one to five copies of the transfecting DNA associated with, and most probably integrated into, cellular DNA sequences. In several transformants, vector-coded gene products for which there was no selection are also synthesized. This suggests that recombinant DNAs containing Ecogpt as a selective marker can be useful for cotransformation of nonselectable genes.

Hydroxyl radical production in body fluids. Roles of metal ions, ascorbate and superoxide.

Abstract: Hydroxyl radical production, detected by ethylene formation from methional, has been investigated in plasma, lymph and synovial fluid. In the presence of added iron-EDTA, addition of either H2O2 or xanthine and xanthine oxidase gave rise to hydroxyl radical formation that in most cases was not superoxide-dependent. The ascorbate already present in the fluid appeared to participate in the reaction. In the absence of added catalyst, the reaction was hardly detectable, the rate being less than 5% of that observed with 1 microM-iron-EDTA added. This implies that the fluids had little if any capacity to catalyse hydroxyl radical production via this mechanism.

Gut bacteria recycle uric acid nitrogen in termites: A strategy for nutrient conservation.

Abstract: Reticulitermes flavipes termites synthesize uric acid via purine-nucleoside phosphorylase (purine-nucleoside: orthophosphate ribosyltransferase, EC 2.4.2.1) and xanthine dehydrogenase (xanthine:NAD+ oxidoreductase, EC 1.2.1.37), but their tissues lack uricase (urate:oxygen oxidoreductase, EC 1.7.3.3) or any other enzyme that degrades uric acid. Nevertheless, uricolysis occurs in termites, but as an anaerobic process mediated by hindgut bacteria. 14C-Tracer experiments showed that termites transport uric acid from the site of synthesis and storage (fat body tissue) to the site of degradation (hindgut microbiota) via Malpighian tubules. Moveover, [1,3-15N]uric acid dissimilated by gut bacteria in vivo leads to assimilation of 15N into termite tissues. NH3, a product of uricolysis, is a potential N source for termites, either directly via glutamine synthetase [L-glutamate:ammonia ligase (ADP-forming), EC 6.3.1.2] activity of fat body tissue or indirectly through microbe assimilation. Symbiotic recycling of uric acid N appears to be important to N conservation in these oligonitrotrophic insects.

The Metabolism of Purine and Pyrimidine Nucleotides in Rat Cortex During Insulin‐Induced Hypoglycemia and Recovery

Abstract: Brains of paralysed rats with insulin-induced hypoglycemia were frozen in situ after spontaneous EEG activity had been absent for 5 or 15 min (“coma”). Recovery (30 min) was achieved in a different group of rats by administering glucose after a 30-min coma period. Purine and pyrimidine nucleotides, nucleosides and free bases were determined in the cortical extracts by high pressure liquid chromatography (HPLC). The ATP values obtained with the HPLC method were in excellent agreement with those obtained using standard enzymatic/fluorometric techniques, while values for ADP and AMP obtained with the HPLC method were significantly lower. Comatose animals showed a severe (40-80%) reduction in the concentrations of all nucleoside triphosphates (ATP. GTP, UTP and CTP) and a simultaneous increase in the concentrations of all nucleoside di- and monophosphates, including that of IMP. The adenine nucleotide pool size decreased to 50% of control level. The concentrations of the nucleosides adenosine, inosine, and uridine increased 50- to 250-fold, while the concentrations of the purine bases, xanthine and hypoxanthine, rose 2- and 30-fold, respectively. There were no increases in the concentrations of adenine, guanine, or xanthosine. Following glucose administration there was a partial (ATP, UTP and CTP) or almost complete (GTP) recovery of the nucleoside triphosphate levels. During recovery, the levels of nucleosidc di- and monophosphates and of adenosine decreased to values close to control; the rise in the inosine level was only partially reversed, and the concentrations of hypoxanthine and xanthine rose further. The adenine nucleotide pool size was only partially restored (to 67% of control value). The adenine nucleotide pool size was not increased by i.p. injection of adenosine or adenine under control condition, or during the posthypoglycemic recovery period.

Cytokinin interaction with free radical metabolism and senescence: Effects on endogenous lipoxygenase and purine oxidation

Abstract: A typical system comprising xanthine-xanthine oxidase, which produces superoxide free radicals, significantly increased endogenous levels of the senescence-associated lipoxygenase enzyme while cytokinin reversed this effect. It is suggested that in its interaction with free radicals cytokinin may have a dual effect: a) it may inhibit purine oxidation by the formation of a 2,8 dihydroxy purine which lowers the substrate affinity of xanthine oxidase; b) it may act as a direct free radical scavenger by virtue of H abstraction from the α-carbon atom in the amine bond.

Inhibition of separated forms of cyclic nucleotide phosphodiesterase from pig coronary arteries by 1,3-disubstituted and 1,3,8-trisubstituted xanthines.

Abstract: A series of xanthines with varied substituents in the 1, 3, and 8 positions were prepared in an attempt to understand the structure--activity relationship for alkylxanthines as inhibitors of two different forms of cyclic nucleotide phosphodiesterase. Polar substituents on the 1 or 3 position of the xanthine reduced the potency of the xanthines to inhibit both the calmodulin-sensitive and the "cyclic AMP specific" forms of phosphodiesterase. Polar substituents on the 8 position of the xanthine, other than a carboxylic acid, increased the potency to inhibit the calmodulin-sensitive form of phosphodiesterase, if they were capable of donating electrons to the xanthine nucleus. On the other hand, any substituent in the 8 position larger than H reduced the potency of the xanthines to inhibit the cyclic AMP specific form of phosphodiesterase. Topographical maps of the active sites of the two forms of phosphodiesterase are presented in summary.

Adenine, the precursor of nucleic acids in intestinal cells unable to synthesize purines de novo.

Abstract: The capacity of rat intestinal epithelial cells for de novo purine synthesis and reutilization was studied in vitro with isolated cells and in vivo in functionally hepatectomized rats. De novo purine synthesis and purine reutilization were measured as the rate of incorporation of [14C]-glycine or [14C]-adenine, respectively, into the adenine and guanine pools of the intestinal cells. Isolated intestinal epithelial cells incubated with labeled purine or glycine incorporated only labeled adenine. Labeled glycine, guanine, hypoxanthine or xanthine were not incorporated into cellular adenine or guanine. Labeled glycine, given intravenously to functionally hepatectomized rats, was not incorporated into the adenine and guanine of intestinal villus or crypt cells. Labeled glycine was readily incorporated into hepatic adenine and guanine pools of a sham-operated rat. The failure of glycine to be incorporated into intestinal cells in vitro or in vivo demonstrated that these cells lack de novo purine synthesis. Since adenine was readily incorporated into the adenine pool of these cells, we believe that adenine, which is synthesized in the liver or supplied in the diet, was an important precursor for the nucleic acid synthesis in this study.

Purine metabolism in the protozoan parasite Eimeria tenella

Abstract: Crude extracts of the oocysts of Eimeria tenella, a protozoan parasite of the coccidium family that develops inside the caecal epithelial cells of infected chickens, do not incorporate glycine or formate into purine nucleotides; this suggests lack of capability for de novo purine synthesis by the parasite. The extracts, however, contain high levels of activity of the purine salvage enzymes: hypoxanthine, guanine, xanthine, and adenine phosphoribosyltransferases and adenosine kinase. The absence of AMP deaminase from the parasite indicates that E. tenella cannot convert AMP to GMP; the latter thus has to be supplied by the hypoxanthine, xanthine, or guanine phosphoribosyltransferase of the parasite. These three activities are associated with one enzyme (HXGPRTase), which has been purified to near homogeneity in high yield (71-80%) in a single step by GMP-agarose affinity column chromatography. The size of the enzyme subunit is estimated to be 23,000 daltons by NaDodSO4 gel electrophoresis. Kinetic studies suggest differences in purine substrate specificity between E. tenella HXGPRTase and chicken liver HGPRTase. Allopurinol preferentially inhibits the parasite enzyme by competing with hypoxanthine; a Ki approximately 22 microM.

Recovery of nucleotide levels after cell injury.

Abstract: The major pathway of purine catabolism in mouse kidney during ischemia occurs through IMP, inosine, hypoxanthine, and xanthine. Short periods of ischemia (reversible cell injury) allow a rapid return of the energy charge to control values and a rapid return of ATP and GTP to values of 60–70% of control ATP and GTP then slowly return to control levels over the next 24 h. Long periods of ischemia (irreversible cell injury; ischemic times longer than 1 h) allow a gradual return of the energy charge to control levels. ATP, GTP, or total adenine or guanine nucleotides do not return to control levels even after 24 h of reinfusion under these circumstances. We conclude that irreversibly injured kidney cells retain the ability to phosphorylate purine nucleotides, but lose the ability to restore the concentrations of the purine nucleotides to control values.

Postchemotherapy Purine Excretion in Lymphoma Patients Receiving Allopurinol

Abstract: The urinary excretion of hypoxanthine, xanthine, and uric acid was measured prior to and following chemotherapy in 11 patients with rapidly growing chemotherapy-sensitive lymphomas who were receiving concomitant allopurinol therapy. Mean maximal total daily urinary excretions of these purines post-chemotherapy were: uric acid, 807 mg/day; hypoxanthine, 343 mg/day; and xanthine, 638 mg/day. The mean maximal postchemotherapy urinary concentrations of uric acid, hypoxanthine, and xanthine were 288, 115, and 179 mg/liter, respectively. Mean total daily urinary excretion of uric acid, hypoxanthine, and xanthine rose 2.2-, 6.6-, and 6.9-fold, respectively, following initiation of antineoplastic therapy. Although standard doses of allopurinol did not prevent a post-chemotherapy increase in the excretion of uric acid or hypoxanthine, the urinary concentrations of both compounds remained below their solubility in urine at pH 7 in all 11 patients studied. However, the urinary concentration of xanthine exceeded its solubility in urine at pH 7 in six of the 11 patients. In three of the six patients whose urinary xanthine concentration exceeded its solubility in urine, transient renal failure developed in association with the increased excretion of xanthine. These studies indicate that, despite the use of conventional doses of allopurinol, the urinary excretion of uric acid may still increase following massive tumor lysis, and urinary excretion of xanthine can increase to concentrations potentially causing xanthine nephropathy.

Effect of NADH on hypoxanthine hydroxylation by native NAD+-dependent xanthine oxidoreductase of rat liver, and the possible biological role of this effect.

Abstract: The course of the reaction sequence hypoxanthine leads to xanthine leads to uric acid, catalysed by the NAD+-dependent activity of xanthine oxidoreductase, was investigated under conditions either of immediate oxidation of the NADH formed or of NADH accumulation. The enzymic preparation was obtained from rat liver, and purified 75-fold (as compared with the 25000 g supernatant) on a 5′-AMP-Sepharose 4B column; in this preparation the NAD+-dependent activity accounted for 100% of total xanthine oxidoreductase activity. A spectrophotometric method was developed for continuous measurements of changes in the concentrations of the three purines involved. The time course as well as the effects of the concentrations of enzyme and of hypoxanthine were examined. NADH produced by the enzyme lowered its activity by 50%, resulting in xanthine accumulation and in decreases of uric acid formation and of hypoxanthine utilization. The inhibition of the Xanthine oxidoreductase NAD+-dependent activity by NADH is discussed as a possible factor in the regulation of IMP biosynthesis by the ‘de novo’ pathway or (from unchanged hypoxanthine) by ther salvage pathway.

Purification and characterization of the intestinal promoter of iron(3+)-transferrin formation

Abstract: The nonceruloplasmin enzyme located in the intestinal mucosa which promotes the incorporation of iron into transferrin has been resolved into a small, heat-stable component and a heat-labile protein component The small, heat-stable component was purified from the high-speed supernatant of intestinal mucosal homogenates by ion-exchange chromatography and gel filtration and identified as xanthine The heat labile protein component was purified from the high-speed supernatant of intestinal mucosal homogenates by heat treatment, gel filtration, and ion-exchange chromatography The physical, spectral, and kinetic properties of the heat-labile protein component strongly suggest that it is xanthine oxidase By promotion of the oxidation and incorporation of iron into transferrin, intestinal xanthine oxidase could perform a similar function in iron absorption as ceruloplasmin serves in the mobilization of iron from liver stores

Purification and properties of urate oxidase from Streptomyces cyanogenus.

Abstract: Urate oxidase [EC 1.7.3.3] was purified to homogeneity from cell-free extracts of a strain of Streptomyces cyanogenus. The enzyme had a molecular weight of 100,000 and consisted of three subunits each with a molecular weight of 32,000. The isoelectric point was at pH 4.0. No evidence was found for the involvement of copper, iron or coenzymes in the urate oxidase reaction. The enzyme was most active at pH 8 and at 35 degrees C, and was stable between pH 6 and 11 (35 degrees C, 1 h) and below 50 degrees C (pH 7.8, 10 min). The enzyme was inhibited by cyanide and sulfhydryl reagents, but only slightly by heavy metal ions and chelating agents. The activity was inhibited by xanthine and 2-hydroxypurine. The enzyme was found not to be inhibited by high concentrations of uric acid when the activity was assayed in terms of hydrogen peroxide formation. Urea and racemic allantoin were formed from uric acid by the enzyme reaction in phosphate buffer, and urea and other ninhydrin-positive materials in borate buffer.

Superoxide, xanthine oxidase and platelet reactions: further studies on mechanisms by which oxidants influence platelets.

Abstract: In the course of studying the effects on platelets of the oxidant species superoxide (O2.-), O2.- was generated by the interaction of xanthine oxidase plus xanthine. Surprisingly, gel-filtered platelets, when exposed to xanthine oxidase in the absence of xanthine substrate, were found to generate superoxide (O2.-), as determined by the reduction of added cytochrome c and by the inhibition of this reduction in the presence of superoxide dismutase. In addition to generating O2.-, the xanthine oxidase-treated platelets display both aggregation and evidence of the release reaction. This xanthine oxidase induced aggregation is not inhibited by the addition of either superoxide dismutase or cytochrome c, suggesting that it is due to either a further metabolite of O2.0, or that O2.- itself exerts no important direct effect on platelet function under these experimental conditions. The ability of C2.- to modulate platelet reactions in vivo or in vitro remains in doubt, and xanthine oxidase is an unsuitable source of O2.- in platelet studies because of its own effects on platelets.

Purification and characterization of hypoxanthine-guanine phosphoribosyltransferase from Saccharomyces cerevisiae.

Abstract: Hypoxanthine-guanine phosphoribosyltransferase (HPRT) was purified 12 000-fold to homogeneity from yeast by a three-step procedure including acid precipitation, anion-exchange chromatography, and guanosine 5' -monophosphate affinity chromatography. The enzyme is a dimer consisting of two, probably identical, subunits of Mr 29 500. The enzyme recognized hypoxanthine and guanine, but not adenine or xanthine, as substrates. An antiserum against both native and denatured enzyme has been raised and shown to be specific for the enzyme. The antiserum has no affinity for Chinese hamster or human HPRT but does recognize subunits of yeast HPRT as well as some cyanogen bromide fragments of the enzyme.

Molekulare und kinetische Charakterisierung der Xanthin-Dehydrogenase aus dem phototrophen Bakterium Rhodopseudomonas capsulata / Molecular and Kinetic Characterization of Xanthine Dehydrogenase from the Phototrophic Bacterium Rhodopseudomonas capsulata

Abstract: The structural and kinetic properties of xanthine dehydrogenase (EC 1.2.1.37) from the facultative phototrophic bacterium Rhodopseudomonas capsulata were studied. The enzyme was fully induced when hypoxanthine or xanthine, but less effectively when uric acid served as nitrogen source during growth. The enzyme was purified about 2300-fold from cells grown photosynthetically with hypoxanthine as N-source by using ammoniumsulfate precipitation, gel filtration, ion-exchange and affinity chromatography. The molecular weight as determined by gel filtration throug Sephacryl S-300 was 345000. Subunit analysis by sodium dodecyl sulfate gel electrophoresis suggested a composition of four identical subunits with a molecular weight of 84000. The enzyme contained 2 flavin, 2 molybdenum and 8 iron-sulfur groups per mol. The turnover number with hypoxanthine and NAD as substrates was 12000 min⁻¹. Hypoxanthine, 1-methylhypoxanthine, 8-azahypoxanthine, xanthine, 1-methylxanthine, 2-hydroxypurine, 6,8-di-hydroxypurine, 5-azacytosine and 5-azauracil served as electron donors. The most effective electron acceptor was NAD. The kinetic constants (Kₘ) were (in μm): 52.5 (hypoxanthine); 32.5 (xanthine) and 61.2 (NAD). Various purine compounds inhibited the enzyme competitively in respect to hypoxanthine as substrate. Although reduction of uric acid to xanthine was not detected by using purified enzyme preparations, in vitro-experiments with ¹⁴C-labelled uric acid indicated that the enzyme xanthine dehydrogenase participates in uric acid degradation in Rps. capsulata. According to their electrophoretic mobilities, the xanthine dehydrogenases isolated from hypoxanthine-and uric acid-grown cells were identical.

Purification and properties of xanthine oxidase from Enterobacter cloacae.

Abstract: Enterobacter cloacae KY 3074 grown in a medium containing xanthine, hypoxanthine, guanine, or their nucleosides and nucleotides produced xanthine oxidase. The purified enzyme preparation showed a major protein band and a few minor bands in acrylamide gel electrophoresis. Molecular oxygen was the most effective electron acceptor. Ferricyanide and 2,6-dichlorophenolindophenol also served as electron acceptors, but NAD and NADP did not. Xanthine and hypoxanthine were good substrates, and guanine was also an effective substrate. The activity was inhibited by Ag2+, Cu2+, PCMB, and ascorbate. The spectrum of the Enterobacter enzyme resembled that of some known xanthine oxidizing enzymes, and this suggests a similarity in the prosthetic groups of these enzymes. The molecular weight of the native enzyme and subunit was 128,000 and 69,000, respectively.

Mode of reactions between xanthine oxidase and aromatic nitro compounds.

Abstract: The electron transfer mechanism in the reduction of aromatic nitro compounds by xanthine oxidase was investigated using methyl p-nitrobenzoate and p-nitroacetophenone as substrates. Methyl p-nitrobenzoate was reduced by both one-electron and more than twoelectron transfer mechanisms in the enzyme-electron donor system. When NADH was used as an electron donor, the ratio of one-electron flux to the total electron flux (the summation of one-electron and more than two-electron fluxes) was dependent on pH of the medium, but not on the concentration of the nitro compounds. The reverse was the case when the electron donor was xanthine. Additional experiments showed that methyl p-nitrobenzoate or p-nitroacetophenone was reduced to the corresponding hydroxylamino compounds and amino compounds by xanthine oxidase supplemented with xanthine or NADH. In these cases, the pattern of formation of the reduction products was dependent on the enzyme activity. The present study strongly suggested that the reduction of aromatic nitro compounds by xanthine oxidase proceeds through the four-electron and six-electron transfer mechanisms as well as the one-electron transfer mechanism.