Inhibition of mammalian xanthine oxidase by folate compounds and amethopterin

We have examined the effects of folate compounds and the folate analog amethopterin (methotrexate) as inhibitors of mammalian xanthine oxidase and have found that they offer potent inhibition of the enzyme. We have compared the inhibitory potency of folic acid and its coenzyme derivative tetrahydrofolic acid to that of allopurinol, a known inhibitor of xanthine oxidase, and have demonstrated that folic acid and tetrahydrofolic acid are severalfold more potent than allopurinol as inhibitors of xanthine oxidase. Comparative inhibition constants calculated were 5.0 X 10(-7) M for folic acid. 1.25 X 10(-6) M for tetrahydrofolic acid, and 4.88 X 10(-6) M for allopurinol. Incubation of xanthine oxidase with folic acid at a concentration of 10(-6) M abolished 94% of the enzymic activity within 1 min of incubation with the enzyme. At the same concentration, allopurinol was almost ineffective as an inhibitor of xanthine oxidase. The substrate xanthine protected the enzyme against total inhibition by folic acid. Reversibility of the enzymic inhibition by folic acid was demonstrated. Folic acid-inactivated enzyme was totally regenerated either by filtration through Sephadex G-200 or by precipitation with ammonium sulfate. 2-Amino-4-hydroxypteridine was a poor substrate for the enzyme but a potent inhibitor for the oxidation of xanthine by the enzyme. The inhibition constant calculated was 1.50 X 10(-6) M. In the presence of an excess of xanthine oxidase, neither folic acid nor tetrahydrofolic acid and allopurinol exhibited any change in intensity of their absorbance or in the wavelength of their maximal absorbance that might have been suggestive of substrate utility. The folate analog amethopterin was also determined a potent inhibitor of mammalian xanthine oxidase. The inhibition constant calculated was 3.0 X 10(-5) M.     

Mechanism of interaction between milk xanthine oxidase and p-chloromercuribenzoate. Properties of the purified enzyme]

Two types of complexes are formed during the interaction of xanthine oxidase with p-chloromercurybenzoate (pCMB). The reversible inactive complex (presumably of absorption nature) is formed practically instantaneously and competitively with regard to the substrate (Ki=6,2 . 10(-8) M) in 0,05 M phosphate buffer (pH 7,8, 25 degrees) and does not involve the fast-reacting SH-groups of the enzyme. Reactivation of xanthine oxidase is observed during prolonged incubation of the inactive complex at 0 degrees; it is associated with the interaction between pCMB and the fact-reacting SH-groups. This interaction results in a dissociation of the inactive complex. The blocking of the slow-reacting SH-groups is accompanied by an irreversible loss of the xanthine oxidase activity. The enzyme modification by blocking of 10 fast-reacting SH-groups does not involve the Fe-S clusters, but results in local changes in the enzyme conformation. This is manifested in a 2-fold increase of Km and the rate constants of proteolysis of the modified xanthine oxidase as compared to the native enzyme. The rate constants of proteolysis by trypsin for the native and modified enzymes in 0,05 M phosphate buffer (pH 7,8; 37 degrees) are 3,7 . 10(-3) min-1 and 7,0 . 10(-3) min-1, respectively; those for chymotrypsin in the same buffer (30 degrees) are 1,5 . 10(-2) min-1 and 6,0 . 10(-2) min-1, respectively.     

Effect of benzodiazepines on the activity of AMP deaminase and adenosine deaminase in rat brain tissue in vivo

Phenazepam (5 mg/200 g) and seduxen (3 mg/200 g) injected intraperitoneally to 184 rats altered AMP-deaminase and adenosine deaminase brain activity. Seduxen was observed to increase AMP-deaminase and adenosine deaminase activity by 89.1% and 32.4%, respectively an hour after the injection. Phenazepam increased the activity of the enzymes by 35.5% and 38.5%, respectively two hours after the injection. The effect is suggested to be due to de novo benzodiazepine-induced enzyme synthesis.     

Correlation between the experimental and clinical effectiveness of benzodiazepines and their affinity for benzodiazepine receptors

The therapeutic effect of 6 benzodiazepine tranquilizers (diazepam, oxazepam, chlordiazepoxide, phenazepam, lorazepam, nitrazepam) was compared to the activity displayed in the most widely used experimental models. The methods of conflict situation, antagonism with thiosemicarbazide and corasole were found to be highly significant for predicting the clinical efficacy of benzodiazepines. The conditioned reflex techniques were shown untenable for estimating the therapeutic action of the tranquilizers. The correlation was discovered between integral clinical tranquilizing effects of benzodiazepines, their experimental activity and affinity to benzodiazepine receptors.     

Effect of alterations in membrane lipid unsaturation on the properties of the insulin receptor of Ehrlich ascites cells

Rat heart ornithine decarboxylate activity from isoproterenol-treated rats was inactivated in vitro by reactive species of oxygen generated by the reaction xanthine/xanthine oxidase. Reduced glutathione, dithiothreitol and superoxide dismutase has a protective effect in homogenates and in partially purified ornithine decarboxylase exposed to the xanthine/xanthine oxidase reaction, while diethyldithiocarbamate, which is an inhibitor of superoxide dismutase, potentiated the damage induced by O2- on enzyme activity. Dithiothreitol at concentrations above 1.25 mM had an inhibitory effect upon supernatant ornithine decarboxylase activity, while at 2.5 mM it was most effective in the recovery of ornithine decarboxylase activity, after the purification of the enzyme by the ammonium sulphate precipitation procedure. The ornithine decarboxylase inactivated by the xanthine/xanthine oxidase reaction showed a higher value of Km and a reduction of Vmax with respect to control activity. The exposure of rats to 100% oxygen for 3 h reduced significantly the isoproterenol-induced heart ornithine decarboxylase activity. The injection with diethyldithiocarbamate 1 h before hyperoxic exposure further reduced heart ornithine decarboxylase activity.     

Interaction of milk xanthine oxidase with folic acid. Inhibition of milk xanthine oxidase by folic acid and separation of the enzyme into two fractions on Sepharose 4B/folate gel

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.     

A simple method for measuring the kinetics of the formation of oxygen radicals

Sodium 2, 6-dichloroindophenol (DCIP Na ) was used to measure the kinetics of the formation of oxygen radicals. The oxygen radicals react with DCIP - , resulting in a decrease in DCIP concentration which is monitored by the decrease in A . A method based on this principle was demonstrated with xanthine/xanthine oxidase giving a K = 4.9x10 M. Cu significantly inhibited the enzyme reaction.     

Dehydrogenase conversion to oxidase and lipid peroxidation in brain after carbon monoxide poisoning.

The conversion of xanthine dehydrogenase to xanthine oxidase and lipid peroxidation were measured in brain from carbon monoxide- (CO) poisoned rats. Sulfhydryl-irreversible xanthine oxidase increased from a control level of 15% to a peak of 36% over the 90 min after CO poisoning, while the conjugated diene level doubled. Reversible xanthine oxidase was 3-6% of the total enzyme activity over this span of time but increased to 31% between 90 and 120 min after poisoning. Overall, reversible and irreversible xanthine oxidase represented 66% of total enzyme activity at 120 min after poisoning. Rats depleted of this enzyme by a tungsten diet and those treated with allopurinol before CO poisoning to inhibit enzyme activity exhibited no lipid peroxidation. Treatment immediately after poisoning with superoxide dismutase or deferoxamine inhibited lipid peroxidation but had no effect on irreversible oxidase formation. Biochemical changes only occurred after removal from CO, and changes could be delayed for hours by continuous exposure to 1,000 ppm CO. These results are consistent with the view that CO-mediated brain injury is a type of postischemic reperfusion phenomenon and indicate that xanthine oxidase-derived reactive oxygen species are responsible for lipid peroxidation.     

Mechanisms of action of malondialdehyde and 4-hydroxynonenal on xanthine oxidoreductase

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.     

Effects of Ca2+ on flavin-linked complex enzymes in mitochondria isolated from eggs and embryos of sea urchin

Mitochondria isolated from sea urchin embryos in early development show almost the same activities of cytochrome c oxidase and flavin-linked complex enzymes, which are estimated by cytochrome c reductases as in those isolated from unfertilized eggs. The activities of these cytochrome c reductases are inhibited by Ca2+ at above 10-5 M more strongly than cytochrome c oxidase. To investigate the changes in intramitochondrial Ca2+ concentration at fertilization, the activity of pyruvate dehydrogenase, another mitochondrial enzyme, was measured. The activity of this enzyme was controlled by phosphorylation and Ca2+-dependent dephosphorylation of the catalytic unit. The enzyme activity increased for 30 min after fertilization, decreased and became close to zero within ~60 min. Then, the activity appreciably increased again after hatching. This seems to reflect changes in the intramitochondrial Ca2+ concentration. The enzyme activity was enhanced by pre-incubation with Ca2+ at concentrations up to 10-5 M but was made quite low at above 10-4 M Ca2+ and 10-3 M adenosine triphosphate. Although the changes in pyruvate dehydrogenase activity observed at fertilization will reflect the changes in the intramitochondrial calcium concentration, the intramitochondrial Ca2+ concentration of unfertilized eggs cannot be estimated from these results because high (> 10-4 M) or low (10-6 M) Ca2+ can inhibit the enzyme. Measurement of respiration of a single egg showed that injection of ethyleneglycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid released the mitochondrial electron transport in the unfertilized egg. The possibility that changes in intramitochondrial calcium concentration occur at fertilization is discussed in relation to activation of both mitochondrial respiration and pyruvate dehydrogenase.     

Xanthine Oxidase Catalyzes the Synthesis of Retinoic Acid

Milk xanthine oxidase (xanthine: oxygen oxidore-ductase; XO; EC 1.1.3.22) was found to catalyze the conversion of retinaldehyde to retinoic acid. The ability of XO to synthesize all trans-retinoic acid efficiently was assessed by its turnover number of 31.56 min^?1, determined at pH 7.0 with 1nM XO and all trans-retinaldehyde varying between 0.05 to 2μM. The determination of both retinoid and purine content in milk was also considered in order to correlate their concentrations with kinetic parameters of retinaldehyde oxidase activity. The velocity of the reaction was dependent on the isomeric form of the substrate, the all trans- and 9-cis-forms being the preferred substrates rather than 13-cis-retinaldehyde. The enzyme was able to oxidize retinaldehyde in the presence of oxygen with NAD or without NAD addition. In this latter condition the catalytic efficiency of the enzyme was higher. The synthesis of retinoic acid was inhibited 87% and 54% by 4μM and 2μM allopurinol respectively and inhibited 48% by 10 μM xanthine in enzyme assays performed at 2μM all trans-retinaldehyde. The K_i value determined for xanthine as an inhibitor of retinaldehyde oxidase activity was 4 μM.     

An amperometric xanthine oxidase enzyme electrode based on hydrogen peroxide electroreduction

A xanthine oxidase enzyme electrode (xanthine oxidase immobilized on electrochemically modified graphite and conveniently coated with gelatine electrode working surface) for quantitative analysis of xanthine is proposed. The detection of thus developed electrochemical system is based on the electroreduction of hydrogen peroxide generated in enzyme layer and offered L-ascorbic and uric acid reducing interference effect on the substrate determination. At a working potential -50 mV (vs. Ag/AgCl) the detection limit of 4.5 microM and the linearity of the amperometric signal up to substrate concentration of about 40 microM were found. At that working potential, the electrode is practically inert towards L-ascorbic- and uric acid present. The response time did not exceed 2 min.     

Inhibitory effects of methylxanthines on the activity of xanthine oxidase

The $\text{in vitro}$ and $\text{in vivo}$ effects of three methylxanthines caffeine, theophylline and theobromine on the activity of the enzyme xanthine oxidase (EC 1.2.3.2.) was investigated with a view to understand their biochemical action. The studies revealed all the three methylxanthines to be inhibitors of the milk xanthine oxidase activity and the inhibition was found to be competitive in nature. The preincubation studies indicated a greater inhibition of the enzyme with the methylxanthines. Excessive amount of the substrate (2.5 × 10^?4M) resulted in progressive inhibition of the enzyme activity. Low concentrations of methylxanthines exerted a definite inhibitory effect on the xanthine oxidase activity at lower substrate concentrations. At higher concentrations of the substrate, the inhibitory effect due to the same concentration of methylxanthines did not produce any added inhibition of the enzyme activity to that produced by the substrate alone. However, added inhibition by high concentrations of methylxanthines was detectable even when the enzyme activity was markedly inhibited by higher concentrations of the substrate. The $\text{in vivo}$ administration of methylxanthines caused a significant inhibition of the xanthine oxidase activity in lungs, kidneys, heart and brain of rats. Consequently, the level of uric acid in the tissues of the drug treated animals was also found to be reduced.     

Association of xanthine oxidase with the bovine milk-fat-globule membrane. Catalytic properties of the free and membrane-bound enzyme

1. The catalytic properties of xanthine oxidase in bovine milk (EC 1.2.3.2) are dependent on the state of the enzyme, i.e. whether free or bound to the fat-globule membrane. Oxidase activity of the membrane-bound enzyme towards NADH is enhanced relative to that towards xanthine. This reflects a change in the relative K(m) values and enables the ratio of xanthine to NADH oxidase activities (X/N) to be used as a parameter for the relative amounts of free and membrane-bound xanthine oxidase in milk fractions. 2. Chromatography of buttermilk on Sepharose 2B yielded an excluded fraction, BM(1), with xanthine oxidase activity. The remaining xanthine oxidase activity was eluted as a single broad peak. This was further resolved on Sephadex G-200 into an excluded fraction, BM(2), and free xanthine oxidase. Fractions BM(1) and BM(2) had X/N values in the range 45-65, which is characteristic of membrane-bound xanthine oxidase. Purified xanthine oxidase has a mean X/N value of 110.3. Addition of fraction BM(1), heated to remove associated enzyme activities, to purified xanthine oxidase progressively enhanced its NADH oxidase activity to a value where its X/N value was characteristic of membrane-bound xanthine oxidase. This was shown to be due to binding of free enzyme to heated fraction BM(1). The binding constant and stoicheiometry were determined. 4. Proteolytic digestion of fraction BM(1) liberated free xanthine oxidase from the fat-globule membrane with a corresponding alteration in X/N value.     

Xanthine oxidase-catalyzed reductive debromination of 6-(bromomethyl)-9H-purine with concomitant covalent modification of the FAD prosthetic group

Bovine milk xanthine oxidase was potently inhibited by 6-(bromomethyl)-9H-purine in a time-dependent process with O2 as the electron acceptor. If the enzyme were assayed with phenazene ethosulfate as an electron acceptor, 6-(bromomethyl)-9H-purine was not an inhibitor. The rate of formation of inhibited enzyme increased with increasing concentrations of 6-(halomethyl)-9H-purine, decreased with increasing concentrations of O2, and increased in the presence of xanthine. The inhibited enzyme regained activity nonactinically at pH 7 with a t1/2 of 31 h. The optical difference spectrum between native enzyme and inhibited enzyme suggested that the enzyme-bound FAD was modified. This conclusion was confirmed by demonstrating that activity was restored to the inhibited enzyme if the enzyme-bound flavin was removed by treatment with CaCl2 and the resulting apoenzyme was reconstituted with FAD. Aerobically, 6-(bromomethyl)-9H-purine was oxidized by the enzyme to a species having a UV spectrum consistent with hydroxylation of the purine ring to form a urate analogue. Anaerobically, the enzyme reduced 6-(bromomethyl)-9H-purine to 6-methylpurine with 1 mol of enzyme being completely inhibited after reduction of 23 mol of 6-(bromomethyl)-9H-purine. Thus, 6-(bromomethyl)-9H-purine was not only oxidized by xanthine oxidase but was also reduced by the enzyme in a reaction that partitioned between formation of 6-methylpurine and inhibition of the enzyme by modification of the enzyme-bound flavin. Similar results were found when 6-(chloromethyl)-9H-purine was the inhibitor.     

Colloidal gold as a biocompatible immobilization matrix suitable for the fabrication of enzyme electrodes by electrodeposition

Glucose oxidase, horseradish peroxidase, xanthine oxidase, and carbonic anhydrase have been adsorbed to colloidal gold sols with good retention of enzymatic activity. Adsorption of xanthine oxidase on colloidal gold did not result in a change in enzymatic activity as determined by active site titration with the stoichiometric inhibitor pterin aldehyde and by measurement of the apparent Michaelis constant (K'(M)). Gold sols with adsorbed glucose oxidase, horseradish peroxidase, and xanthine oxidase have also been electrodeposited onto conducting matrices (platinum gauze and/or glassy carbon) to make enzyme electrodes. These electrodes retained enzymatic activity and, more importantly, gave an electrochemical response to the enzyme substrate in the presence of an appropriate electron transfer mediator. Our results demonstrate the utility of colloidal gold as a biocompatible enzyme imobilization matrix suitable for the fabrication of enzyme electrodes. (c) 1992 John Wiley & Sons, Inc.     

Phosphoserine phosphatase of human brain: Partial purification,characterization, regional distribution,and effect of certain modulators including psychoactive drugs

Phosphoserine phosphatase (PSPase), a cytosolic enzyme has been purified 106 fold from human brain, by employing conventional protein purification techniques. The use of MgCl₂ (10 mM) and chloroform treatment, during purification enabled the removal of non-specific proteins. The final enzyme preparation exhibited a broad pH optimum of 5.6–6.6 and could dephosphorylate bothl andd enantiomers of the phosphoserine, but with different Km values for O-P-L serine (3.6×10^–5M) and O-P-D serine (1×10^–4M). Enzyme activity was found to be specific for phosphoserine, whereas other phosphoesters including phosphothreonine and phosphoproteins such as casein and phosvitin were found to be poor substrates. The enzyme activity was uncompetitively inhibited byl-serine. Further the PSPase activity was inhibited by vanadate, (41%), trifluoperazine (23%), chlorpromazine (34%) at an equimolar concentration of 1 mM, whereas lithium and ethanol did not influence the enzyme activity. Minor tranquilizers such as diazepam and chlordiazepoxide activated the enzyme activity to an extent of 13% and 59% respectively. In addition, species and regionwise heterogeneity was observed with respect to distribution of enzyme activity in six major areas of human, rabbit and rat brains.     

Lysosomal enzyme leakage during the hypoxanthine/xanthine oxidase reaction

Impairment of lysosomal stability due to reactive oxygen species generated during the oxidation of hypoxanthine by xanthine oxidase was studied in rat liver lysosomes isolated in a discontinuous Nycodenz gradient. Production of O2.- and H2O2 during the hypoxanthine/xanthine oxidase reaction occurred for at least 5 min, while lysosomal damage, indicated by the release of N-acetyl-beta-glucosaminidase, occurred within 30 s, there being no further damage to these organelles thereafter. The extent of lysosomal enzyme release increased with increasing xanthine oxidase concentration. Superoxide dismutase and catalase did not prevent lysosomal damage during the hypoxanthine/xanthine oxidase reaction. Lysosomes reduced xanthine oxidase activity, as assessed in terms of O2 consumption, only slightly but substantially inhibited in a competitive manner the O2.- -mediated reduction of cytochrome c. This inhibition was almost completely reversed by potassium cyanide, thus pointing to the presence of a cyanide-sensitive superoxide dismutase in the lysosomal fraction. However, potassium cyanide did not affect the hypoxanthine/xanthine oxidase-mediated lysosomal damage, thus suggesting an inability of the lysosomal superoxide dismutase to protect the organelles. Negligible malondialdehyde formation was observed in the lysosomes either during the hypoxanthine/xanthine oxidase reaction or with different selective experimental approaches known to produce lipid peroxidation in other organelles such as microsomes and mitochondria.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of proteoglycan synthesis by hydrogen peroxide in cultured bovine articular cartilage

Oxygen-derived reactive species, generated enzymatically by the action of xanthine oxidase upon hypoxanthine, significantly inhibit proteoglycan synthesis by cultured bovine articular cartilage (Bates, E.J., Lowther, D.A. and Handley, C.J. (1984) Ann. Rheum. Dis. 43, 462-469). Here we extend these investigations and show, through the use of catalase and the specific iron chelator diethylenetriaminepentaacetic acid, that the active species involved is H2O2 and not the hydroxyl radical. Incubations of cartilage with H2O2 at concentrations of 1 X 10(-4) M and above are also inhibitory to proteoglycan synthesis. Subsequent recovery of the tissue is dependent upon the initial dose of xanthine oxidase or H2O2. Xanthine oxidase at 84 mU per incubation results in a prolonged inhibition of proteoglycan synthesis which is still apparent after 14 days in culture. Lower concentrations of xanthine oxidase (21-66 mU) are inhibitory to proteoglycan synthesis, but the tissue is able to synthesise proteoglycans at near normal rates after 3 days in culture. The inhibition of proteoglycan synthesis by 1 X 10(-4) M H2O2 is completely reversed after 5 days in culture, whereas 1 X 10(-3) M H2O2 results in a more prolonged inhibition. The synthesis of the proteoglycan core protein is inhibited, but the ability of the newly formed proteoglycans to aggregate with hyaluronic acid is unimpaired.     

Lack of conversion of xanthine dehydrogenase to xanthine oxidase during warm renal ischemia

Irreversible transformation of xanthine dehydrogenase (XDH) to xanthine oxidase (XO) during ischemia was determined measuring XDH and total enzyme activity in kidneys before and after 60 min of clamp of the renal pedicle. Tissue levels of adenine nucleotides, xanthine and hypoxanthine were used as indicators of ischemia. After 60 min of clamping, ATP levels decreased by 72% with respect to controls whereas xanthine and hypoxanthine progressively reached tissue concentrations of 732 +/- 49 and 979 +/- 15 nmol.g tissue-1, respectively. Both total and XDH activities in ischemic kidneys (30 +/- 15 and 19 +/- 1 nmol.min-1.g tissue-1) were significantly lower than in controls when expressed on a tissue weight basis. The fraction of enzyme in the XDH form was however unchanged indicating that the reduction of the nucleotide pool is not accompanied by induction of the type-O activity of xanthine oxidase.