Showing papers on "Xanthine published in 1975"

The interaction of bovine erythrocyte superoxide dismutase with hydrogen peroxide: chemiluminescence and peroxidation.

Abstract: Reaction of bovine erythrocyte superoxide dismutase with H2O2 was accompanied by a luminescence whose intensity was a function of the concentration of H2O2 and whose duration was coincident with the inactivation of the enzyme by this reagent. Oxygen, which protected against inactivation, also diminished the luminescence. Several other compounds which prevented the inactivation by H2O2 also modified the luminescence. Thus urate, formate, and triethylamine inhibited luminescence whereas imidazole and xanthine augmented it. These seemingly contrary effects can be explained by assuming that the compounds which protected the enzyme were peroxidized in competition with the sensitive group on the enzyme. The luminescence arises because that group on the enzyme was oxidized to a product in an electronically excited state, which could return to the ground state by emitting light. Imidazole and xanthine gave electronically excited products whose quantum efficiency was greater than that of the group on the enzyme, whereas urate, formate, and triethylamine gave products with much lower luminescent efficiencies. This superoxide dismutase could catalyze the peroxidation of a wide range of compounds, including ferrocytochrome c, luminol, diphenylisobenzofuran, dianisidine, and linoleic acid. In control experiments, boiled enzyme was inactive. This peroxidative activity can lead to unexpected effects when superoxide dismutase is added to H2O2-producing systems, as a probe for the involvement of O2-. Several examples from the literature are cited to illustrate the misinterpretations which this previously unrecognized peroxidative activity can generate.

6 Molybdenum Iron-Sulfur Flauin Hydroxylases and Related Enzymes

Abstract: Publisher Summary This chapter describes the enzymes, xanthine oxidase, xanthine dehydrogenase, aldehyde oxidase, and sulfite oxidase. In addition to the molybdenum hydroxylases, sulfite oxidase is discussed in this chapter, though it contains heme rather than flavin and no iron-sulfur. This is because it contains molybdenum, apparently in a chemical environment within the active center, which is quite like that of the metal in the other enzymes. The term xanthine oxidase is to be taken to refer to the enzyme from milk. The enzymes to be considered are widely distributed. Thus, one or more molybdenum hydroxylases have been found in organisms as different in complexity as man and bacteria. In mammal's oxidation of hypoxanthine to xanthine and of xanthine to uric acid are steps in purine catabolism prior to excretion, uric acid being the end product in primates. Individual humans, genetically deficient in xanthine oxidase, are apparently little the worse for the deficiency. The reduced oxygen derivatives are supposed to be available for coupled oxidation reactions—for example, in drug catabolism. The most obvious objection to this hypothesis is the conclusion, discussed in the chapter, that the dehydrogenase form of the xanthine-oxidizing enzymes is more native than is the oxidase form. It must be conceded that the true role of the enzymes in humans and many other species remains somewhat problematical.

Metabolism of N-methylpurines by a Pseudomonas putida strain isolated by enrichment on caffeine as the sole source of carbon and nitrogen.

Abstract: Pseudomonas putida, strain 40, originally isolated by enrichment on caffeine as the sole source of carbon and nitrogen, has been developed to grow on 0.5% caffeine. The organism will grow on any N-methyl derivative of xanthine containing one or more methyl groups at the 1, 3, or 7 positions. An investigation of the activities of resting cell suspensions and cell-free preparations together with the detection of metabolic intermediates suggest that caffeine is first metabolized by the action of an enzyme which is capable of hydrolytically removing all three methyl groups with the production of methanol and free xanthine. The methanol presumably is oxidized to the final product, CO2, through the sequential action of methanol, formaldehyde, and formate dehydrogenases, which are induced by growth on caffeine. Furthermore, the xanthine would seem to be channeled through conventional pathways of purine degradation through the action of xanthine dehydrogenase and uricase, both induced by growth on caffeine. However, a variety of data suggests that the metabolism of caffeine may be compartmentalized in the cell and metabolized separately from externally added xanthine. Additional studies indicated that the cell is permeable to the methylxanthines. The significance of these findings is discussed.

Biosynthesis of caffeine by tea-leaf extracts. Enzymic formation of theobromine from 7-methylxanthine and of caffeine from theobromine.

Abstract: 1. Extracts prepared from tea leaves with Polyclar AT (insoluble polyvinylpyrrolidine) contained two methyltransferase activities catalysing the transfer of methyl groups from S-adenosylmethionine to 7-methylxanthine, producing theobromine, and to theobromine, producing caffeine. 2. The methyltransferases exhibited the same pH optimum (8.4) and a similar pattern of effects by metal ions, thiol inhibitors and metal-chelating reagents, both for theobromine and caffeine synthesis. Mg2+, Mn2+ and Ca2+ slightly stimulated enzyme activity but they were not essential. Paraxanthine was shown to be most active among methylxanthines, as the methyl acceptor. However, the formation of paraxanthine from 1-methylxanthine was very low and that from 7-methylxanthine was nil, suggesting that the synthesis of caffeine from paraxanthine is of little importance in intact plants. Xanthine, xanthosine, XMP and hypoxanthine were all inactive as methyl acceptors, whereas [2(-14)C]xanthine and [8(-14)C]hypoxanthine were catabolized to allantoin and urea by tea-leaf extracts. The apparent Km values are as follows: 7-methylxanthine, 1.0 times 10(-14)M; theobromine, 1.0 times 10(-3)M; paraxanthine, 0.2 times 10(-3)M; S-adenosylmethionine, 0.25 times 10(-4)M (with each of the three substrates). 3. The results suggest that the pathway for caffeine biosynthesis is as follows: 7-methylxanthine leads to theobromine leads to caffeine. In contrast, it is suggested that theophylline is synthesized from 1-methylxanthine. The methyl groups of the purine ring of caffeine are all derived directly from the methyl group of S-adenosylmethionine. Little is known about the pathways leading to the formation of 7-methylxanthine. 4. A good correlation between caffeine synthesis and shoot formation or growth of tea seedlings was shown, suggesting that the methylating systems in caffeine synthesis are closely associated with purine nucleotide and nucleic acid metabolism in tea plants.

Uptake and supply of purine compounds by the liver.

Abstract: We have analyzed for purine compounds entering and leaving the liver in lightly anesthetized rabbits and rats and for the export of utilizable purine from liver perfused with oxypurine. The in vivo results indicate that roughly 80% of hypoxanthine, xanthine, and urate is removed in a single passage of blood through liver. Conversely, the adenosine concentration of hepatic venous blood is increased 10-fold over portal or arterial levels. When the liver is isolated and perfused with hypoxanthine there is significant release of adenosine, whether measured quantitatively by microbiological assay or qualitatively by analysis of the radioactive purines released from liver that has been prelabeled with [14C]hypoxanthine. These results provide direct evidence for the clearance of hydroxylated purines and the release of utilizable adenine derivatives by liver.

Genetic and metabolic control of the purine catabolic enzymes of Neurospora crasse.

Abstract: Neurospora crassa can utilize various purine bases such as xanthine or uric acid and their catabolic products as a nitrogen source. Four classes of mutants which affect the purine degradative pathway were isolated and studied. Mutants of the aln-1 class specifically lack allantoinase, while alc-1 mutants lack allantoicase. Mutants designated as xdh-1 cannot utilize hypoxanthine as a nitrogen source and are presumed to be deficient in xanthine dehydrogenase activity. A regulatory mutant, amr, was found to have only very low, uninduced levels of uricase, allantoinase, and allantoicase. None of these genes are closely linked to each other. The three initial enzymes involved in the catabolism of uric acid are controlled in a complex manner by both induction and repression. Several lines of evidence indicate that the true inducer of uricase and allantoicase is uric acid. The use of the newly isolated mutant strains made it possible to demonstrate that neither allantoin nor allantoic acid could act as inducers. Furthermore, hypoxanthine itself was shown to be ineffective as an inducer although it can be metabolized to form an inducer. A non-metabolizable analogue of uric acid, 8-azaxanthine, is a gratuitous inducer of these enzymes. Uricase and allantoicase were found to be synthesized coordinately, but they were not coordinately regulated with allantoinase. Both uricase and allantoicase are stable enzymes and do not undergo turnover; nor are they subject to feedback inhibition by ammonia. Allantoinase, however, is quite labile both in vivo and in vitro. This enzyme was found to turnover in vivo in the presence of cycloheximide with a half-life of approximately 20 minutes. The amr (for ammonia regulation) mutant cannot utilize a wide range of compounds, including purines, nitrate, and many amino acids as a nitrogen source and also displays a multiple enzyme loss. The amr gene appears to play a major role in the control of nitrogen metabolism. It is postulated that the amr locus encodes a regulatory protein which is required to activate transcription of the structural genes for a group of related enzymes involved in nitrogen metabolism.

The determination of hypoxanthine and xanthine with a PO2 electrode.

Abstract: Extract: A simple and rapid method for determination of the hypoxanthine and xanthine concentration in plasma and urine is described. The method is based on the principle that oxygen is consumed quantitatively when hypoxanthine and xanthine are oxidized to urate by xanthine oxidase. By using Henry's law a direct measure of the hypoxanthine and xanthine concentration is obtained. The method determines these oxypurines in volumes of 200 μl in concentrations less than 5 μmol/liter in about 5 min. The average precision in the range of 0–50 μmol/liter is 2.6 μmol/liter. Of the added hypoxanthine, 99 102% is recovered in plasma. Even though xanthine oxidase is a rather nonspecific enzyme, experiments show that this method is highly specific during physiologic conditions. Speculation: Other purines which can be metabolized to hypoxanthine can be determined by this method. For instance, inosine, which is metabolized to hypoxanthine by nucleoside phosphorylase in the presence of phosphate, might also be determined according to this method.

Metabolism of xanthine and hypoxanthine in the tea plant (Thea sinensis L.)

Abstract: 1. The metabolism of xanthine and hypoxanthine in excised shoot tips of tea was studied with micromolar amounts of [2(-14)C]xanthine or [8(-14)C]hypoxanthine. Almost all of the radioactive compounds supplied were utilized by tea shoot tips by 30 h after their uptake. 2. The main products of [2(-14)C]xanthine and [8(-14)C]hypoxanthine metabolism in tea shoots were urea, allantoin and allantoic acid. There was also incorporation of the label into theobromine, caffeine and RNA purine nucleotides. 3. The results indicate that tea plants can catabolize purine bases by the same pathways as animals. It is also suggested that tea plants have the ability to snythesize purine nucleotides from glycine by the pathways of purine biosynthesis de novo and from hypoxanthine and xanthine by the pathway of purine salvage. 4. The results of incorporation of more radioactivity from [8(-14)C]hypoxanthine than from [2(-14)C]xanthine into RNA purine nucleotides and caffeine suggest that hypoxanthine is a more effective precursor of caffeine biosynthesis than xanthine. The formation of caffeine from hypoxanthine is a result of nucleotide synthesis via the pathway of purine salvage.

Avian xanthine dehydrogenases. I. Isolation and characterization of the turkey liver enzyme.

Abstract: 1. 1. Xanthine dehydrogenase was isolated from turkey liver in good yield and with specific activity and spectral properties comparable to the best preparations of these enzymes from other sources. 2. 2. The enzyme contains two Mo, two FAD, eight Fe and eight labile sulphides per mol. The molecular weight and molar extinction per equivalent of FAD at 450 nm are 280,000 and 36,550, respectively. 3. 3. The enzyme exhibits a relatively low substrate and electron acceptor specificity. A preliminary investigation of the xanthine: NAD+ oxidoreductase activity shows that “ping-pong” type kinetics obtains only over a limited range of substrate concentration.

Kinetic Studies Dealing with an Immobilized Bienzyme System

Abstract: The binding of enzymes into artificial membranes makes possible a study of the interaction between membrane structure and enzyme kinetics within a simple context. Artificial protein membranes bearing a bienzyme system (xanthine oxidase, uricase) are produced by using a co-crosslinking method. The inhibition of uricase was shown to be dependent not only on the concentration of inhibitor in the bulk solution, but also on the kinetic properties of the membrane-bound enzymes. In the presence of xanthine oxidase inside the structure the uricase inhibition by xanthine is less important than in solution. Under defined conditions the activity was found to be higher in the presence of inhibitor than in its absence. Due to diffusion limitations this specific bienzyme system is more efficient when immobilized inside a membrane than when in solution.

The effects of a new xanthine derivative, 3,7-dimethyl-1(5-oxohexyl)-xanthin (pentoxifylline) on the general and cardiac haemodynamics of the intact animal.

Abstract: A study has been made of the effects of xanthine derivative (pentoxifylline) upon the general and cardiac haemodynamics of intact anaesthetised dogs. The drug increased heart-rate, respiration rate, cardiac output and systemic pressure. It reduced mean pulmonary arterial pressure. Coronary flow was increased for only a few minutes, but coronary sinus O2 content increased throughout the study. Lactate and pyruvate values increased in both artery and coronary sinus, as did the cardiac extraction of these substances. Cardiac extraction of non-esterified fatty acid increased.

3,5-Disubstituted 1,2,4-triazoles, a new class of xanthine oxidase inhibitor

Abstract: 3,5-Bis(4-pyridyl)-1,2,4-triazole (PPT), 3-(4-pyrimidinyl)-5-(4-pyridyl)-1,2,4-triazole (PMPT), and 3-(4-pyridazinyl)-5-(4-pyridyl)-1,2,4-triazole (PZPT) are among the most active competitive inhibitors of xanthine oxidase among a series of 3,5-disubstituted triazoles synthesized for this purpose, inhibition constants being less than 1 times 10(-7) M for each. ED50 values in squirrel monkeys derived from first-order rate constants for the first and rate-limiting step of the sequence, xanthine leads to uric acid leads to allantoin plus CO2, range from 0.04 to 0.08 mg kg-1 orally, with unusually long durations of action attributable to asymmetric distribution of inhibitor within liver and gut as a consequence of enterohepatic recirculation. Sensitivity of rats, dogs, and anthropoid species to these, as to other xanthine oxidase inhibitors, is markedly less than that of the squirrel monkey, but the triazoles are at least an order of magnitude more active than the representative purine analogs tested.

The influx of uric acid and other purines into everted jejunal sacs of the rat and hamster.

Abstract: The in vitro transport of [2-14C]uric acid, [8-14C]hypoxanthine, and [8-14C]xanthine, each dissolved in Krebs–Ringer bicarbonate buffer, was studied with everted jejunal sacs from rat and hamster. No evidence could be obtained for the development of a concentration gradient between the intracellular fluid and the incubation medium or between the sac contents and the incubation medium, for any of the three oxypurines. Inhibitors of active transport, such as anaerobiosis or dinitrophenol, had no significant effect on the rate of transport. A large percentage of hypoxanthine and xanthine was oxidized to uric acid in the sac-wall homogenate, sac contents, and incubation medium during the course of the incubation. This oxidation could be prevented by addition of allopurinol (3 mM) to the incubation medium, but concentration gradients were still not obtained. No active transport mechanism could be demonstrated for uric acid, hypoxanthine, or xanthine in rat or hamster jejunum.

Oxidizing Action of Purine N-Oxide Esters

Abstract: A technique involving O-acetylation of purine N-oxide derivatives in buffered aqueous solutions has permitted studies of the reactivity of many compounds for which the O-acetyl derivatives are not otherwise available. The oxidizing properties of a variety of N-acetoxypurines have been measured through their ability to oxidize iodide ion ot iodine, a reaction which is representative of a more general oxidizing ability. Those esters that oxidize iodide ion also catalyze the autoxidation of sulfite, a property characteristic of radicals. The same esters also oxidize cysteine to cysteic acid and tryptophan, tyrosine, and uric acid to yet uncharacterized products. Their oxidizing reactivity was compared with the ability of the same esters to react as electrophiles in another assay that measured the rate of formation of pyridine substitution products. The sulfate ester of 3-hydroxyxanthine has been synthesized. Its reactivity is qualitatively the same as that of 3-acetoxyxanthine but proceeds at a higher rate. Syntheses of S-(8-xanthyl)-N-acetylcysteine, 8-(2-hydroxyethylthio)xanthine, and 1-methyl-8-mehtylmercaptoguanine are also described.

Guanosine nucleotide precursor for flavinogenesis of eremothecium ashbyii

Abstract: The purine precursor in the riboflavin biosynthetic pathway in Eremothecium ashbyii was examined using a guanine analogue, 8-azaguanine, with non-growing cell systems. 1. Riboflavin formation in the culture filtrate was determined at 0, 5, 10 and 20 hr after start of the incubation of the non-growing cells in the presence of xanthine or 8-azaguanine (1mM, respectively). At 20 hr of incubation, the addition of xanthine stimulated riboflavin formation by 36% and the addition of 8-azaguanine inhibited the formation by 57%. 2. Acid soluble nucleotide pools in the cells were followed at 0, 5, 10 and 20 hr of the incubation period in the presence of xanthine or 8-azaguanine by means of anion exchange column chromatography. The result showed that the GTP pool changed markedly despite the fact that the adenosine nucleotide pool was almost constant irrespective of the presence or absence of these purines till 10 hr of incubation. Put, the decrease of the former was overcome in part by the addition of flavinogenic xanthine. Furthermore, the total amounts of GTP and guanosine accumulated in cells in the presence of 8-azaguanine reached the maximum already at 5 hr, attaining a level twice as much as the GTP contents of the control. 3. The role of guanosine nucleotide pool in riboflavin formation was further examined using 8-azaguanine. In this experiment the drug was added to the suspension of non-growing cells at 3 hr or 6 hr after the incubation was started and the reaction was continued till the 12th hr. A more clear-cut correlationship between riboflavin formation and guanosine nucleotide pool was observed by this experiment. The guanosine nucleotide pool (consisting of GMP, GDP and GTP) increased simultaneously with the inhibition of riboflavin formation. Of the guanosine nucleotides pools, the GMP pool increased 2.7 times above normal upon the addition of 8-azaguanine during the incubation for 6 hr and 5.3 fold for 9 hr. While, the GTP pool increased 1.9 fold above normal for 6 hrs' incubation in the supplementation of this drug but decreased to one-half of the normal at the incubation period of 9 hr. In these cases, the decreased amounts of GTP were equal to the increased amounts of GMP during the incubation periods of 6 hr and 9 hr in the presence of added 8-azaguanine. 4. The above results suggest strongly that GTP is an immediate precursor of riboflavin in the form of nucleotide.

Synthesis of uric acid in isolated normothermic perfused mongrel and Dalmatian dog kidneys.

Abstract: Renal synthesis of uric acid (urate) in the dog was demonstrated by use of two isolated kidney preparations during pulsatile perfusion at 37 degrees C with artificial perfusate or with a plasma fraction. During perfusion of mongrel and Dalmatian dog kidneys with 0.5 mg of [14C]xanthine per 100 ml, a mean of 24.3 and 25.4 mug of [14C]urate per minute, respectively, entered either urine or perfusate after its synthesis in the isolated kidney. Approximately 6.2% of the combined extracellular radiolabeled urate formed in the isolated mongrel kidney was excreted in the urine and 15.7% in the urine of isolated Dalmatian dog kidneys. Recirculating the perfusate without the kidney did not convert any radioactively labeled xanthine to urate and therefore the radioactively labeled urate appearing in the urine and recycled perfusate must have been formed in the renal parenchyma. Renal synthesis of urate was blocked by the xanthine oxidase inhibitor allopurinol. In the presence of allopurinol, [14C]xanthine was excreted unchanged into the urine.

Which of caffeine's chemical relatives are able to evoke contractures in mammalian heart?

Abstract: At concentrations between 0.5 mM and 10 mM caffeine, theophylline, theobromine, imidazole, 2-methyl imidazole, 5-amino-4-imidazole carboxamide, naphazoline, antazoline, and tolazoline in normal MacEwen solution induce contractures in rat and guinea pig myocardium. 9-Methylxanthine, 1,3,9-trimethylxanthine, hypoxanthine, or 2-imidazolidone, when similarly applied, do not. These observations, and similar results from frog heart, can be interpreted assuming that an unsaturated position 9 nitrogen in a xanthine molecule or in the equivalent position of an imidazole group is necessary for the release of calcium into the sarcoplasm to be initiated and therefore for a contraction to develop. The site of action of these compounds is likely to be the sarcoplasmic reticulum.

Xanthine, hypoxanthine and muscle pain. Histochemical and biochemical observations.

Abstract: A suspected case of xanthine oxidase deficiency has been further investigated. The patient complained of arthralgia and myalgia. Further studies included histochemical and ultramicroscopic analysis of muscle sarcoplasmic reticulum, and biochemical studies. High levels of xanthine and hypoxanthine were found, while uric acid was absent in the muscle extracts.

Kinetics and mechanism of the 3H to 1H in C(8)H groups of purine derivatives

Abstract: The pH-dependence of the 3H to 1H exchange between water and C(8)H groups of purine, adenine, 9-methyladenine, 7-methyladenine, hypoxanthine, guanine, xanthine as well as C(2)H groups of imidazole and benzimidazole was studied. It was shown that within the pH-ranges, where the majority of molecules under study are non-ionized, the values of observed rate constant (kobs) do not depend on pH. Beyond these ranges the values of k(obs) are increased or decreased depending on the type of ionizaiton of the compound under study in appropriate pH range. The observed pH dependence of the 3H to 1H exchange is in a good quantitative agreement with ylide mechanism of the exchange reaction. According to this mechanism the 3H--1H exchange takes place in N(7)-protonated forms of the purine derivatives and in zwitterions with positive charge on N(7). The ylide mechanism of the exchange reaction is also suggested by the fact that the true exchange rate constants (k+) of protonated forms of the studied compounds, calculated from the values of k(ods), rises linearly with the increase of their protonation constant (Ka1)--the tenfold increase of Ka1 leads to about four-fold rise of k+. The knowledge of 1h to 3H exchange mechanism in C(8)H groups of purine derivatives allows to estimate alterations of reactivity of the purine residues in polynucleotides and nucleic acids depending on their conformation.

Avian xanthine dehydrogenases. II. Accumulation of intermediates during the oxidation of purines by the turkey liver enzyme: calculation of the concentration of each component during the reaction.

Abstract: 1. 1. The transient accumulation of intermediates in the conversion of purine to uric acid by turkey liver xanthine dehydrogenase was followed by ultraviolet differential spectrophotometry. 2. 2. A method of calculating the concentration of each component in such reaction mixtures is presented. 3. 3. The pathway of oxidation was shown to be purine → hypoxanthine → xanthine → uric acid . 4. 4. The xanthine/uric acid ratio in incubation mixtures was shown to be a function of reaction time, enzyme concentration and initial hypoanthine concentration. 5. 5. The conversion of xanthine to uric acid obeys pseudo first-order kinetics. No evidence for dismutation of xanthine at high xanthine concentrations was found.