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

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.     

A histochemical method for detecting xanthine oxidase activity in the liver

A histochemical method is described for localizing xanthine oxidase--the key enzyme in the purine catabolic pathway. The above method is based on the reduction of p-Nitroblue tetrazolium during hypoxanthine enzymatic oxidation, phenazine methosulfate being an intermediate electron acceptor. The patterns of the reaction product distribution suggest that the Kupffer cells and the sinusoidal endothelium possess the highest xanthine oxidase activity.     

Substrate Orientation and Catalytic Specificity in the Action of Xanthine Oxidase: THE SEQUENTIAL HYDROXYLATION OF HYPOXANTHINE TO URIC ACID*

Xanthine oxidase is a molybdenum-containing enzyme catalyzing the hydroxylation of a sp²-hybridized carbon in a broad range of aromatic heterocycles and aldehydes. Crystal structures of the bovine enzyme in complex with the physiological substrate hypoxanthine at 1.8 Å resolution and the chemotherapeutic agent 6-mercaptopurine at 2.6 Å resolution have been determined, showing in each case two alternate orientations of substrate in the two active sites of the crystallographic asymmetric unit. One orientation is such that it is expected to yield hydroxylation at C-2 of substrate, yielding xanthine. The other suggests hydroxylation at C-8 to give 6,8-dihydroxypurine, a putative product not previously thought to be generated by the enzyme. Kinetic experiments demonstrate that >98% of hypoxanthine is hydroxylated at C-2 rather than C-8, indicating that the second crystallographically observed orientation is significantly less catalytically effective than the former. Theoretical calculations suggest that enzyme selectivity for the C-2 over C-8 of hypoxanthine is largely due to differences in the intrinsic reactivity of the two sites. For the orientation of hypoxanthine with C-2 proximal to the molybdenum center, the disposition of substrate in the active site is such that Arg⁸⁸⁰ and Glu⁸⁰², previous shown to be catalytically important for the conversion of xanthine to uric acid, play similar roles in hydroxylation at C-2 as at C-8. Contrary to the literature, we find that 6,8-dihydroxypurine is effectively converted to uric acid by xanthine oxidase.     

Uric acid as electronic acceptor of chicken liver's XDH (author's transl)]

Uric acid seems to act as an electronic acceptor in the dehydrogenation of hypoxanthine catalyzed by chicken liver's xanthinedehydrogenase (XDH). Oxidation was observed in crude homogenates under anaerobic conditions, although dialyzed homogenates or purified hepatic XDH also induce a similar action either in aerobic or anaerobic conditions. The reaction pH optimum is about 6.0. Xanthine appears to be the only inhibited product of the reaction when its concentration is greater than 1 X 10(-4) M. When hypoxanthine and uric acid concentrations exceed 2 X 10(-3) M and 1 X 10(-4) M, respectively, they induce inhibition by substrate. Purine is a fairly good substrate of XDH when uric acid acts as acceptor. Allopurinol inhibits hypoxanthine oxidation by uric acid in the presence of XDH. XDH also catalyzes the dismutation of xanthine to hypoxanthine and uric acid.     

On the mechanism of action of xanthine oxidase. Evidence in support of an oxo transfer mechanism in the molybdenum-containing hydroxylases

The mechanism of action of xanthine oxidase has been investigated using single-turnover experiments in an effort to determine the primary source of the oxygen atom incorporated into product in the course of catalysis. It is found from mass spectroscopic analysis of the uric acid generated in these experiments that when 16O-labeled enzyme in [18O]H2O is reacted with substoichiometric amounts of xanthine (under conditions where no enzyme molecule is likely to react with more than one substrate molecule), the uric acid isolated from the reaction mixture contains 16O at position 8 of the purine ring. Conversely, when 18O-labeled enzyme in [16O]H2O is exposed to substoichiometric xanthine, 18O is incorporated into the product uric acid. These results strongly support a variety of chemical studies with model molybdenum complexes suggesting that the oxygen atom of the Mo = O group known to be present at the active site of xanthine oxidase is transferred to product in the course of catalysis. The mechanistic implications of the present work are discussed.     

Comparison of xanthine:NAD+ oxidoreductase from liver of two uricotelic species: hen Gallus gallus and snake Natrix natrix

1. Kinetic properties of xanthine:NAD+ oxidoreductase from liver of two uricotelic species of vertebrates (hen Gallus gallus and snake Natrix natrix) are compared. 2. Hen enzyme is saturated by hypoxanthine and xanthine at higher concentrations than the snake enzyme. For both species the enzyme-saturating concentration and hydroxylation rate of hypoxanthine are higher than those of xanthine, and the rate of uric acid production in the hypoxanthine----xanthine----uric acid reaction sequence is independent of the initial hypoxanthine concentration. 3. Km's for xanthine are the same, but Km for NAD+ of the hen enzyme is approximately 5-fold lower. The enzyme from both species is inhibited by NADH only slightly and at high non-physiological concentrations.     

Biochemical and structural characterization of the hypoxanthine-guanine-xanthine phosphoribosyltransferase from Pyrococcus horikoshii

The 6-oxopurine phosphoribosyltransferase (HPRT, EC 2.4.2.8) from the hyperthermophile Pyrococcus horikoshii was expressed in Escherichia coli and purified. Steady-state kinetic studies indicated that the enzyme is able to use hypoxanthine, guanine and xanthine. The first two substrates showed similar catalytic efficiencies, and xanthine presented a much lower value (around 20 times lower), but the catalytic constant was comparable to that of hypoxanthine. The enzyme was not able to bind to GMP-agarose, but was able to bind the other reverse reaction substrate, inorganic pyrophosphate, with low affinity (K(d) of 4.7+/-0.1 mM). Dynamic light scattering and analytical gel filtration suggested that the enzyme exists as a homohexamer in solution.     

Ultrastructural localization of xanthine oxidase activity in the digestive tract of the rat

Summary Precise localization of xanthine oxidase activity might elucidate physiological functions of the enzyme, which have not been established so far. Because xanthine oxidase is sensitive to chemical (aldehyde) fixation, we have localized its activity in unfixed cryostat sections of rat duodenum, oesophagus and tongue mounted on a semipermeable membrane. Previous studies had shown that this procedure enables the exact localization of activities of peroxisomal oxidases with maintenance of acceptable ultrastructure. Moreover, leakage and/or diffusion of enzyme molecules was prevented with this method. The incubation medium to detect xanthine oxidase activity contained hypoxanthine as substrate and cerium ions as capturing agent for hydrogen peroxide. After incubation, reaction product in the sections was either visualized for light microscopy or sections were fixed immediately and processed for electron microscopy. At the ultrastructural level, crystalline reaction product specifically formed by xanthine oxidase activity was found to be present in the cytoplasmic matrix of enterocytes and goblet cells and in mucus of duodenum. Moderate activity was found in the cytoplasm of apical cell layers of epithelia of oesophagus and tongue, with highest activity in the cornified layer. Moreover, large amounts of reaction product were found to surround bacteria present between cell remnants of the cornified layer of the oesophagus. Many bacteria surrounded by the enzyme showed signs of destruction and/or cell death. The intracellular localization of xanthine oxidase activity in the cytoplasm of epithelial cells as well as the extracellular localization suggest that the enzyme plays a role in the lumen of the digestive tract, for instance in the defence against microorganisms.     

Soybean nodule xanthine dehydrogenase: a kinetic study

Xanthine dehydrogenase was purified from soybean nodules and the kinetic properties were studied at pH 7.5. Km values of 5.0 +/- 0.6 and 12.5 +/- 2.5 microM were obtained for xanthine and NAD+, respectively. The pattern of substrate dependence suggested a Ping-Pong mechanism. Reaction with hypoxanthine gave Km's of 52 +/- 3 and 20 +/- 2.5 microM for hypoxanthine and NAD+, respectively. The Vmax for this reaction was twice that for the xanthine-dependent reaction. The pH dependence of Vmax gave a pKa of 7.6 +/- 0.1 for either xanthine or hypoxanthine oxidation. In addition the Km for xanthine had a pKa of 7.5 consistent with the protonated form of xanthine being the true substrate. Km for hypoxanthine varied only 2.5-fold between pH 6 and 10.7. Product inhibition studies were carried out with urate and NADH. Both products gave mixed inhibition with respect to both substrates. Xanthine dehydrogenase was able to use APAD+ as an electron acceptor for xanthine oxidation, with a Km at pH 7.5 of 21.2 +/- 2.5 microM and Vmax the same as that obtained with NAD+. Reduction of APAD+ by NADH was also catalyzed by xanthine dehydrogenase with a Km of 102 +/- 15 microM; Vmax was approximately 2.5 times that for the xanthine-dependent reaction, and was independent of pH between 6 and 9. Reaction with group-specific reagents indicated the possibility of an essential histidyl group. A thiol-modifying reagent did not cause inactivation of the enzyme. A role for the histidyl side chain in catalysis is proposed.     

Inhibition of xanthine oxidase by uric acid and its influence on superoxide radical production.

The inhibition of xanthine oxidase by its reaction product, uric acid, was studied by steady state kinetic analysis. Uric acid behaved as an uncompetitive inhibitor of xanthine oxidase with respect to the reducing substrate, xanthine. Under 50 microM xanthine and 210 microM oxygen, the apparent K(i) for uric acid was 70 microM. Uric acid-mediated xanthine oxidase inhibition also caused an increase in the percentage of univalent reoxidation of the enzyme (superoxide radical production). Steady-state rate equations derived by the King-Altman method support the formation of an abortive-inhibitory enzyme-uric acid complex (dead-end product inhibition). Alternatively, inhibition could also depend on the reversibility of the classical ping-pong mechanism present in xanthine oxidase-catalyzed reactions.     

Studies on the mechanism of action of xanthine oxidase

Recent studies of the reaction mechanism of the molybdenum-containing enzyme xanthine oxidase are presented. The pH-dependence of both the steady-state and rapid reaction kinetics of the enzyme exhibits is bell-shaped, with pK(a)s for the acid and alkaline limbs of 6.6 and 7.4, respectively. These are assigned to ionizations of an active site base and substrate, respectively, with the implication that enzyme acts on the neutral rather than monoanionic form of the purine substrate. A computational study provides evidence that in the course of the reaction tautomerization of substrate occurs, with a proton moving from N-3 to N-9 in the course of the reaction - enzyme facilitation of this tautomerization may contribute as much as 24 kcal/mol in transition state stabilization for the reaction. Electron spin echo (ESEEM) and electron-nuclear double resonance (ENDOR) studies of the so-called "very rapid" Mo(V) intermediate of the reaction, the latter work using a newly synthesized form of the substrate 2-hydroxy-6-methylpurine that has been selectively isotopically labeled at C-8, indicates that product is bound to the molybdenum of the active site in a simple, end-on fashion, consistent with a reaction mechanism involving nucleophilic attack of a (deprotonated) Mo-OH on the C-8 position of substrate. A kinetic study using a series of purines has failed to identify a correlation between the one-electron reduction potential for substrate and catalytic effectiveness, indicating that a reaction mechanism initiated by one-electron, outer-sphere electron transfer is unlikely. Finally, a consideration of the active site structure in the context of the above work suggests specific amino acid residues to target for site-directed mutagenesis studies. Preliminary experiments with two such mutants are entirely consistent with the proposed catalytic roles of two active site glutamate residues.     

Xanthine:NAD+ oxidoreductase in the liver of grass snake Natrix natrix

The enzyme hydroxylating oxypurines in the liver of grass snake (Natrix natrix, Colubridae) was found to be a stable xanthine:NAD+ oxidoreductase (EC 1.2.1.37). The Michaelis constants for NAD+ and xanthine amounted to 14.4 and 12.3 microM, respectively. The enzyme affinity to hypoxanthine is lower than that to xanthine, but the former substrate is hydroxylated faster than the latter. The enzyme is only slowly and slightly (up to 22%) inhibited by NADH accumulating during xanthine hydroxylation. The above data and the time-course of hypoxanthine----xanthine----uric acid hydroxylation indicated that the kinetic properties of the snake liver enzyme provide in this uricotelic animal fast elimination of superfluous nitrogen derived from protein catabolism.     

T lymphocyte killing by a xanthine-oxidase-containing immunotoxin

Summary We report on the preparation of an immunotoxin consisting of xanthine oxidase, a free-radicalproducing enzyme, covalently linked to an anti-CD3 monoclonal antibody. The immunotoxin retained both enzymic and immunological properties and its toxicity to target cells (a) was greater than that of the free enzyme, (b) was proportional to the enzyme concentration, and (c) was reduced either in the absence of hypoxanthine or by an excess of free anti-CD3 monoclonal antibody. The cytotoxicity and selectivity of the hypoxanthine/conjugated xanthine oxidase system were potentiated by the addition of chelated iron and by washing away the unbound immunotoxin prior to the addition of substrate. The same system was not toxic to bone marrow progenitor cells. A possible use of this immunotoxin for the ex vivo purging of organs to be transplanted from T lymphocytes, to avoid the graft-versus-host reaction, is suggested.     

Substrate inhibition in a human variant of hypoxanthine-guanine phosphoribosyltransferase

Hypoxanthine-guanine phosphoribosyltransferase from a young man with purine overproduction and decreased purine salvage in fibroblast cultures was found to have low activity at concentrations of purine substrates at which the enzyme from normal individuals showed near maximal activity. The low enzyme activity was not associated with changes in the values of the Km(app) and Vmax(app) for any of the enzyme substrates. However, the enzyme activity was susceptible to substrate inhibition by hypoxanthine and guanine. The values obtained for the true Km, true Vmax, and true Ki for hypoxanthine were 26 +/- 10 microM, 1761 +/- 382 microunits/mg of protein, and 80 +/- 20 microM, respectively. The pattern of the substrate inhibition, as seen on a plot of 1/v versus hypoxanthine concentration, was characteristic of that associated with the formation of a dead-end complex between the inhibitory substrate and an enzyme form with which it normally does not react. The nature of this enzyme form and that of the dead-end complex was determined from double inhibition experiments, which indicated that hypoxanthine interacted with an enzyme-PPi intermediate to form an enzyme-hypoxanthine-PPi dead-end complex. The trapping of the enzyme in this inactive form explains the low activity at high purine base concentrations. Further information as to the nature of the reaction mechanism was obtained from plots of the reciprocal of enzyme activity versus the reciprocal of PP-ribose-P concentration at different fixed hypoxanthine concentrations. A pattern characteristic of uncompetitive substrate inhibition was obtained. This is indicative of an ordered sequential binding of substrates on the enzyme; PP-ribose-P binding before hypoxanthine. Thus, the variant enzyme showed an ordered sequential reaction mechanism, with the inhibitory substrate forming a dead-end complex with an enzyme-PPi intermediate.     

Substrate inhibition of xanthine oxidase and its influence on superoxide radical production.

The influence of substrate inhibition on xanthine oxidase-intramolecular electron transport was studied by steady-state kinetic analysis. Experiments with hypoxanthine and xanthine up to 900 microM indicated an inhibition pattern which fitted an equation of the general form nu 0 = nu max . [S]/(Km + a[S] + b[S]2/Ki). Univalent electron flux to oxygen was favored at substrate concentrations above 50 microM. This augmentation of univalent flux percentage that appeared at a high substrate concentration was greater for hypoxanthine that xanthine and at pH 8.3 than at 9.5. Our results support a mechanism of inhibition in which a substrate-reduced enzyme, non-productive Michaelis complex was formed. It is possible that this non-productive complex favored the univalent pathway of enzyme reoxidation (superoxide production) by increasing the midpoint redox potential of the molybdenum active site.     

The function of subcellular fractions in the oxidation of glutathione in rat liver homogenate

1. The aerobic loss of GSH added to the supernatant fraction from rat liver is much increased by including the microsome fraction, which both inhibits the concurrent reduction of the GSSG formed and also augments the net oxidation rate. 2. Oxidation occurs with a mixture of dialysed supernatant and a protein-free filtrate; the latter is replaceable by hypoxanthine and the former by xanthine oxidase, whereas fractions lacking this enzyme give no oxidation. 3. In all these instances augmentation occurs with microsomes, with fractions having urate oxidase activity and with the purified enzyme; uric acid and microsomes alone also support the oxidation. 4. Evidence implicating additional protein factors is discussed. 5. It is suggested that GSH oxidation by homogenate is linked through glutathione peroxidase to the reaction of endogenous substrate with supernatant xanthine oxidase and of the uric acid formed with peroxisomal urate oxidase.     

Substrate orientation in xanthine oxidase: crystal structure of enzyme in reaction with 2-hydroxy-6-methylpurine

Xanthine oxidoreductase catalyzes the final two steps of purine catabolism and is involved in a variety of pathological states ranging from hyperuricemia to ischemia-reperfusion injury. The human enzyme is expressed primarily in its dehydrogenase form utilizing NAD+ as the final electron acceptor from the enzyme's flavin site but can exist as an oxidase that utilizes O2 for this purpose. Central to an understanding of the enzyme's function is knowledge of purine substrate orientation in the enzyme's molybdenum-containing active site. We report here the crystal structure of xanthine oxidase, trapped at the stage of a critical intermediate in the course of reaction with the slow substrate 2-hydroxy-6-methylpurine at 2.3A. This is the first crystal structure of a reaction intermediate with a purine substrate that is hydroxylated at its C8 position as is xanthine and confirms the structure predicted to occur in the course of the presently favored reaction mechanism. The structure also corroborates recent work suggesting that 2-hydroxy-6-methylpurine orients in the active site with its C2 carbonyl group interacting with Arg-880 and extends our hypothesis that xanthine binds opposite this orientation, with its C6 carbonyl positioned to interact with Arg-880 in stabilizing the MoV transition state.     

Purine utilisation, de novo synthesis and degradation in mouse preimplantation embryos.

The importance of de novo purine synthesis as opposed to the reutilisation of metabolites by salvage pathways, and the nature of the excretory product(s) of purine degradation, have been examined in cultured preimplantation mouse embryos. In the presence of azaserine and mycophenolic acid, which inhibit de novo purine synthesis, embryo cleavage was blocked prior to compaction, the precise stages at which this occurred depended on whether the cultures were established on day 1 or day 2 after fertilisation, and indicated that salvage pathways were insufficient to fulfil the demand for nucleotides during early preimplantation development. The end-product of purine degradation appeared to be xanthine, which was excreted in very small amounts on days 1, 2 and 3, with a pronounced rise from the early to late blastocyst. Uric acid formation or excretion could not be detected. Exogenous hypoxanthine and adenine, which partially inhibited development, were taken up by the embryos and converted to xanthine, most probably by salvage pathways, since the enzyme xanthine oxidase, which converts hypoxanthine directly to xanthine and then to uric acid, could not be detected. Exogenous guanine had little effect on development and was also converted to xanthine, but in this case, the conversion was probably in a single step, via the enzyme guanase.     

Purification and characterization of Escherichia coli xanthine-guanine phosphoribosyltransferase produced by plasmid pSV2gpt

The enzyme xanthine-guanine phosphoribosyltransferase from Escherichia coli cells harboring the plasmid pSV2gpt has been purified 30-fold to near homogeneity by single-step GMP-agarose affinity chromatography. It has a Km value of 2.5, 42 and 182 microM for the substrates guanine, xanthine and hypoxanthine, respectively, with guanine being the most preferred substrate. The enzyme exhibits a Km value of 38.5 microM for PRib-PP with guanine as second substrate and of 100 microM when xanthine is used as the second substrate. It is markedly inhibited by 6-thioguanine, GMP and to a lesser extent by some other purine analogues. Thioguanine has been found to be the most potent inhibitor. The subunit molecular weight of xanthine-guanine phosphoribosyltransferase was determined to be 19 000. The in situ activity assay on a nondenaturing polyacrylamide gel electrophoresis gel has indicated that a second E. coli phosphoribosyltransferase preferentially uses hypoxanthine as opposed to guanine as a substrate, and it does not use xanthine.     

焦磷酸显色法检测PCR产物及酶活性

聚合酶链反应（polymerase chain reaction, PCR）是分子生物学领域的一项具有划时代意义的技术,但定量PCR产物或测定能生成焦磷酸的酶活性仍需要新技术的发展。本文提供了一种PCR产物定性及定量检测方法(Color PCR kit)及其用途;该方法通过焦磷酸(pyrophosphate,PPi)显色测定PCR的副产物PPi。利用试剂盒中的试剂与PPi反应,最终生成物为甲暨（formazan）,呈红色。根据显色现象(红色)判断PCR的阳性结果,由目测实现PCR的定性检测;或通过测定490 nm 处的吸光度值定量检测PCR过程中生成的副产物PPi的含量,定量检测PCR 产物的生成量;目测情况下Color PCR kit可检测到2.5 ng水平的PCR产物,用紫外分光光度计可检测到低限为1 pg;Color PCR kit法比琼脂糖凝胶电泳检测法（低限为4 ng）灵敏。Color PCR kit还可用于连接酶和转移酶活性的测定。