Weak O2 binding and strong H2O2 binding at the non-heme diiron center of trypanosome alternative oxidase

Alternative oxidase (AOX) catalyzes the four-electron reduction of dioxygen to water as an additional terminal oxidase, and the catalytic reaction is critical for the parasite to survive in its bloodstream form. Recently, the X-ray crystal structure of trypanosome alternative oxidase (TAO) complexed with ferulenol was reported and the molecular structure of the non-heme diiron center was determined. The binding of O₂ was a unique side-on type compared to other iron proteins. In order to characterize the O₂ binding state of TAO, the O₂ binding states were searched at a quantum mechanics/molecular mechanics (QM/MM) theoretical level in the present study. We found that the most stable O₂ binding state is the end-on type, and the binding states of the side-on type are higher in energy. Based on the binding energies and electronic structure analyses, O₂ binds very weakly to the TAO iron center (ΔE =6.7 kcal mol^?1) in the electronic state of Fe(II)…OO, not in the suggested charge transferred state such as the superoxide state (Fe(III)OO· ^–) as seen in hemerythrin. Coordination of other ligands such as water, Cl^?, CN^?, CO, N₃^? and H₂O₂ was also examined, and H₂O₂ was found to bind most strongly to the Fe(II) site by ΔE = 14.0 kcal mol^?1. This was confirmed experimentally through the measurement of ubiquinol oxidase activity of TAO and Cryptosporidium parvum AOX which was found to be inhibited by H₂O₂ in a dose-dependent and reversible manner.     

Formation of reactive 1-nitropyrene metabolites by lung microsomes and isolated lung cells

The metabolism and activation of 1-nitropyrene (1-NP) to reactive intermediates by lung microsomes and isolated lung cells was studied. Mutagenicity of 1-NP metabolites was assayed in Salmonella typhimurium TA98NR, a strain lacking a major component of nitroreductase activity. In the presence of NADPH, microsomes from rabbit, rat and hamster lung metabolized 1-NP to mutagenic products to a similar degree. Pretreatment with a mixture of polychlorinated biphenyls (PCB) decreased the formation of mutagenic metabolites by rabbit lung microsomes, but did not affect the production of mutagens by rat or hamster lung microsomes. ³H-1-NP was metabolized to covalently bound protein products at a rate of 82 and 10 pmol/mg by rabbit and hamster lung microsomes, respectively, whereas no binding was detected in rat lung microsomes. PCB-pretreatment increased covalent protein binding of ³ H-1-NP in lung microsomes from hamster and rat, but decreased the binding in rabbit lung microsomes. High performance liquid chromatography analysis indicated that ³H-1-NP was readily converted to ring-hydroxylated products by rabbit and hamster lung microsomes; the rate was much lower with rat lung microsomes. ³H-1-NP was activated to metabolites that covalently bound to protein in isolated rabbit lung cells, with the following rates being observed: Clara cells > lung digest > type II cells. In contrast, covalent protein binding in cells isolated from rat lung was very low. 1-NP was not activated to products mutagenic for S. typhimurium TA 98 N R when co-incubated with cells isolated either from rabbit or rat lung.Abbreviations 1-AP 1-aminopyrene - DMSO dimethyl sulfoxide - EGTA ethylene glycol-bis(ß-aminoethyl ether) - EM electron microscopy - HEPES N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid - HPBS HEPES-phosphate-buffered-saline - HPLC high performance liquid chromatography - NBT nitroblue tetrazolium - 1-NP 1-nitropyrene - 1-NP-4,5-diol trans-4,5-dihydro-4,5-dihydroxy-1-nitropyrene - 1-NP-9,10-diol trans-9,10-dihydro-9,10-dihydroxy-1-nitropyrene - 1-NP-4,5-oxide 1-nitropyrene-4,5-oxide - 1-NP-9,10-oxide 1-nitropyrene-9,10-oxide - 3-OH-1-NP 3-hydroxy-1-nitropyrene - 6-/8-OH-1-NP a mixture of 6- and 8-hydroxy-1-nitropyrene - PBS phosphate-buffered saline - PCB a mixture of polychlorinated biphenyls (Aroclor 1254) - TLC thin layer chromatography     

High-affinity iron binding by xanthine oxidase

Equilibrium dialysis studies on competitive binding of ⁵⁹FeCl₃ to xanthine oxidase and citrate or ATP have been carried out. Iron binding to the enzyme was observed in the presence of 0.1 mM of either chelator, suggesting that xanthine oxidase is likely to have iron bound in many in vitro experimental systems and raising the possibility that it may be able to compete for intracellular chelatable iron. One high-affinity-binding site per monomer was found, with an affinity constant of 5 × 10¹² M⁻¹. The significance of this iron as a Fenton reaction catalyst is discussed.     

Biodegradation of 1-nitropyrene

The metabolism of ¹⁴C-labeled 1-nitropyrene in microcosms containing nonsterile estuarine sediments, and in cultures of a Mycobacterium sp. previously isolated from oil-contaminated sediments was investigated. Although mineralization of 1-nitropyrene by pure cultures of the Mycobacterium sp. totaled only 12.3% after 10 days of incubation, over 80% of the ethyl acetate extractable ¹⁴C-labeled compounds consisted of 1-nitropyrene metabolites. High pressure liquid chromatographic analysis of 1-nitropyrene degradation products indicated that two major metabolites were formed. They were identified as 1-nitropyrene cis-9,10-and 4,5-dihydrodiols, based on their UV-visible, mass and NMR spectra. Time course studies in microcosms showed that 1-nitropyrene was degraded slowly under aerobic and anaerobic conditions in estuarine sediments. Less than 1% had been converted to ¹⁴CO₂ after 8 weeks of aerobic incubation. The addition of 1-nitropyrene to anaerobic sediments resulted in no ¹⁴CO₂ evolution; however, the nitro group of 1-nitropyrene was reduced to form 1-aminopyrene. Although the mineralization of 1-nitropyrene in sediments was slow, the Mycobacterium sp. metabolized 1-nitropyrene in pure culture. This bacterium appears promising for the bioremediation of this ubiquitous pollutant in contaminated waste.Abbreviations DEP Direct exposure probe - HPLC high pressure liquid chromatography - GC/MS gas chromatography/mass spectrometry - Nitro-PAHS nitropolycyclic aromatic hydrocarbons - TLC thin-layer chromatography - UV ultraviolet     

Nature of the phytochrome effect on the binding of glycolate oxidase to peroxisomes in vitro

The attachment of glycolate oxidase to the peroxisomal fraction derived from etiolated barley leaves (Hordeum vulgare L. cr. Dvir) is affected by light. The effect of red irradiation is reversed by subsequent far-red irradiation, indicating the involvement of phytochrome. This phytochrome effect is assumed to be related to phytochrome binding. Indeed, prevention by filipin (1.2·10^-6 mol g^-1 f wt) or cholesterol of phytochrome binding to membranes abolishes the effect of light on the interaction between glycolate oxidase and the peroxisomal fraction. Glycolate oxidase binding is affected by addition of quasi-ionophores such as gramicidin and filipin at a concentration of 0.6·10^-3 mol g^-1 f wt. This fact indicates that peroxisome-glycolate oxidase interaction may be affected by membrane potential. Since both ion transport and membrane potential are known to be affected by phytochrome, it is proposed that phytochrome acts in the light-induced modulation of glycolate oxidase attachment as a quasi-ionophore.Abbreviations GO glycolate oxidase - Pr and Pfr phytochrome forms absorbing in red and far-red, respectively - R and F red and far-red irradiation - Cumulative 20 Kp 20,000 g pellet obtained by centrifugation of the crude extract - 1 Kp 1,000 g pellet - 20 Kp 20,000 g pellet, obtained by centrifugation of 1 Kp supernatant - 1 Kp, 20 Kp and cumulative 20 Kp pellets obtained after density centrifugation through a sucrose cushion     

Functional binding of cardiolipin to cytochromec oxidase

Bovine cytochromec oxidase usually contains 3–4 mol of tightly bound cardiolipin per cytochromeaa ₃ complex. At least two of these cardiolipins are required for full electron transport activity. Without the tightly bound cardiolipin, cytochromec oxidase has only 40–50% of its original activity when assayed in detergents that support activity, e.g., dodecyl maltoside. By measuring the restoration of electron transport activity, functional binding constants for cardiolipin and a number of cardiolipin analogues have been evaluated (K _d,app=1 µM for cardiolipin). These binding constants agree reasonably well with direct measurement of the binding using [¹⁴C]-acetyl-cardiolipin (K _d <0.1 µM) when the enzyme is solubilized with Triton X-100. These data are discussed in relationship to the wealth of data that is known about the association of cardiolipin with cytochromec oxidase and the other mitochrondrial electron transport complexes and transporters.     

Peroxide binding to the type 3 site in Rhus vernicifera laccase depleted of type 2 copper

The interaction between hydrogen peroxide and oxidized $\text{Rhus}\text{vernicifera}$ laccase from which the type 2 copper has been removed, was investigated. For that end, the circular dichroic spectrum of the modified enzyme has been measured in the presence of increasing concentrations of hydrogen peroxide. The characteristic band observed upon binding peroxide to native laccase is also observed for the type 2 copper depleted enzyme. However, there are several quantitative differences in the latter one. First, the intensity is lower and band width is larger. Secondly, from the titrations, it becomes apparent that the affinity for H₂O₂ is markedly lower than that of the native enzyme. While the affinity for the native enzyme is higher than 10⁸ M^?1, it decreases to 1·10⁴ M^?1 for the type 2 depleted enzyme.     

The binding of Mn2+ and ADP to myosin

The metal ion requirement of myosin-ADP binding was investigated by use of Mn²⁺. Mn²⁺ binds to two sets of noninteracting sites on myosin which are characterized by affinity constants of 10⁶ and 10³, M⁻¹ at 0.016 M KCl concentration. The maximum number of sites is 2 for the high affinity and 20–25 for the low affinity set. Binding of Mn²⁺ to the high affinity sites increases the affinity of ADP binding to myosin. F-actin inhibits ADP binding (Kiely, B., and Martonosi, A., Biochim. Biophys. Acta 172: 158–170 [1969]), but even at F-actin concentrations much higher than that required to saturate the actin binding sites of myosin or its proteolytic fragments, significant ADP binding remained. The actin insensitive portion of ADP binding was inhibited by 10⁻⁴ M inorganic pyrophosphate or ATP. The results are discussed on the basis of a model in which actin and ADP bind to myosin at distinct but interacting sites.     

An endothelin type A receptor-expressing cell to characterize endothelin-1 binding and screen antagonist

Endothelin-1 (ET-1) induces contraction of vascular smooth muscle through binding to endothelin type A receptor (ET_AR). COS-7 cells stably expressing high levels of the ET_AR were established (designated COS-7(ET_AR)). The COS-7(ET_AR) cell bound [¹²⁵I]ET-1 with a K_d of 932 ± 161 pM and a B_max of 74 ± 13 fmol/2 × 10⁵ cells. [¹²⁵I]ET-1 binding was inhibited by ET-1 and the ET_AR antagonist BQ-610, but not by the endothelin type B receptor (ET_BR) antagonist BQ-788. In clones expressing two ET_AR mutants containing D46N or R53Q substitutions in the first extracellular domain of the receptor, [¹²⁵I]ET-1 binding activity was dramatically reduced. This suggests that these single amino acid substitutions alter the three-dimensional structure of the ligand-binding domain of the ET_AR. Using COS-7(ET_AR) cell, we showed that Ca²⁺ or Mg²⁺ was essential for ET-1 binding to the ET_AR and that ET-1 treatment induced postreceptor signaling, that is, intracellular accumulation of cyclic AMP (cAMP) and Ca²⁺ mobilization. The COS-7(ET_AR) established in this study will be a useful tool for screening ET-1 antagonists for treating hypertension.     

Compensatory binding of an asparagine residue to the coordination-unsaturated type I Cu center in bilirubin oxidase mutants

Met467, the axial ligand to type I Cu in a multicopper oxidase, Myrothecium verrucaria bilirubin oxidase was substituted with a non-coordinating Phe and Leu to transform the spectral and magnetic properties and oxidase activities of the enzyme into those of fungal laccases, but the mutated type I Cu center showed properties characteristic of phytocyanins, blue copper proteins with an axial coordination of Gln, due to compensatory binding of the distal Asn459 as evidenced by a double mutation.     

The covalent binding of 3-methylindole metabolites to bovine tissue

Tritiated 3-methylindole (3MI) was administered intravenously to calves. Total and covalent bound radioactivity were measured in different tissues. Pulmonary tissue showed the highest concentration of covalent bound radioactivity. (G-³H) or (methyl-¹⁴C) 3MI became covalently bound to microsomal protein when incubated with bovine lung microsomes. This covalent binding was dependent on time, temperature, oxygen and NADPH and was inhibited by SKF-525A, cytochrome c, a carbon monoxide enriched atmosphere and cysteine. The microsomal enzyme system catalysing covalent binding of 3MI has the classical characteristics of a cytochrome P-450 dependent mixed function oxidase. Metabolic activation of 3MI to a highly electrophilic intermediate, may be fundamental in the pathogenesis of 3MI induced pulmonary damage.     

The effects of reversible freezing inactivation and inhibitor binding on redox properties of l-amino-acid oxidase

We measured the redox potentials of frozen inactivated l-amino-acid oxidase (l-amino-acid:oxygen oxidoreductase (deaminating), EC 1.4.3.2) and inhibitor-bound (anthranilic acid) enzyme, and compared these redox properties to those of active l-amino-acid oxidase and benzoate-bound d-amino-acid oxidase (EC 1.4.3.3), respectively. The redox properties of the inactive enzyme are similar to the properties of free flavin; the potential is within 0.015 V of free flavin and no radical stabilization is seen. This corresponds to the loss of most interactions between apoprotein and flavin. In contrast, the anthranilic acid lowers the amount of radical stabilized from 85% to 35%. The potentials are still 0.150 V positive of free flavin, indicating that in the presence of inhibitor, many flavin-protein interactions remain intact. The difference between this behavior and that of d-amino-acid oxidase bound to benzoate, where the amount of radical declined from 95% to 5%, is explained on the basis of the relative tightness of binding of apoprotein to FAD. d-Amino-acid oxidase apoprotein has a relatively low K_a (10⁶) for FAD, and benzoate has a relatively high K_a (10⁵) for the enzyme. Therefore, the binding of benzoate increases the tightness of FAD binding to apo-d-amino-acid oxidase (10¹¹), indicating significant changes in flavin-protein interactions. In contrast, apo-l-amino-acid oxidase binds flavin tightly (the K_a is greater than 10⁷) and the enzyme binds to anthranilate much less tightly, with a K_a of 10³. The l-amino-acid oxidase apoprotein binding to FAD is tight initially, and the binding of anthranilate changes it only slightly. Therefore, redox studies indicate that the ability of a flavoprotein to be regulated may be influenced by the strength of the interaction of flavin with the apoprotein, as well as the strength of interaction of the substrate or activator.     

In-vitro auxin binding to particulate cell fractions from corn coleoptiles

Summary When low concentrations (e.g. 10^-6 M) of labelled 3-indoleacetic acid (¹⁴C-IAA) or -naphthaleneacetic acid (¹⁴C-NAA) are added in vitro to homogenates of corn coleoptiles, radioactivity is reversibly bound to pelletable particles. From the saturation kinetics of the binding it is possible to estimate an apparent K _M between 10^-6 M and 10^-5 M and a concentration of specific sites of 10^-7–10^-6 M per tissue volume.The binding is auxin-specific. Among many compounds tested, only auxins and such auxin analogues that are known to interact directly with auxin in transport and/or growth were found to interfere with this binding. For instance, the growth-active d-dichlorophenoxyisopropionic acid at 10^-4 M inhibits ¹⁴C-NAA binding more than the less active l-isomer.The auxin-binding fractions are practically free of DNA and cytochrome-C oxidase and contain binding sites for 1-naphthylphthalamic acid. The results are discussed in context with the hyothesis—derived mainly from physiological data—that auxin receptors are localized at the plasma membrane.     

High affinity binding site for (+) amphetamine in rat hypothalamus: Fact or artefact?

We studied the binding of (+)³H-amphetamine to membrane sites from rat hypothalamus. Our experiments demonstrate the possible existence of artefactual results in previous studies due to the inadequate use of a filtration technique. In addition, it seems that monoamine oxidase A might be a component of the complex mixture of binding sites which are recognized by (+)³H-amphetamine since harmaline and monoamine oxidase A selective inhibitors are good displacers of this ligand.     

Resonance coherent anti-Stokes Raman scattering (CARS) spectra of flavin adenine dinucleotide,riboflavin binding protein and glucose oxidase

Coherent anti-Stokes Raman scattering spectra, in resonance with the isoalloxazine visible electronic transition, have been obtained down to 300 cm^?1 for flavin adenine dinucleotide, riboflavin binding protein and glucose oxidase, in H₂O and D₂O. Several isoalloxazine vibrational modes can be identified by analogy with those of uracil. Of particular interest is a band at ～1255 cm^?1 in H₂O, which is replaced by another at ～1295 cm^?1, in D₂O. The H₂O band appears to be a sensitive monitor of H-bonding of the N3 isoalloxazine proton to a protein acceptor group. It shifts down by 10 cm^?1 in riboflavin binding protein, and disappears altogether in glucose oxidase. Other band shifts, of 3–5 cm^?1, are similar for the two flavoproteins, and may reflect environmental changes between aqueous solution and the protein binding pockets.     

Lysine binding to activated human platelets and its similarity to fibrinogen binding

Platelet surface glycoproteins IIb-IIIa are considered to function as the binding site for fibrinogen. Fibrinogen binding is essential for platelet aggregation and several amines have been shown to inhibit this binding. The present study compares the binding properties of ¹²⁵I-fibrinogen and [³H]lysine with platelets activated by the Ca²⁺ ionophore A23187. Many lines of similarities in the binding properties are apparent; however, several differences were also found. The similarities are listed below and the differences are pointed out in parentheses. (a) Marked enhancement by platelet activation; (b) deficiency of binding by thrombasthenic platelets lacking the glycoproteins IIb-IIIa; (c) saturability (fibrinogen binding approaches saturation at more than 12 μM, within 10 min; lysine binding at more than 100 mM within 1 min); (d) Ca²⁺-dependence (at 1 mM Ca²⁺ lysine binding is minute and fibrinogen binding is half-saturated); (e) reversibility; the binding achieved within 10 min is exchangeable; dissociation depends upon time and external ligand concentration; (f) inhibition by the oligoamines His-Lys and Lys₄; (g) inhibition by serum from a thrombasthenic patient who developed anti-glycoproteins IIb-IIIa antibodies; (h) specificity; alanine neither binds to activated platelets nor inhibits fibrinogen binding; it thus appears that the lysine which associates with activated platelets is mostly bound onto the surface of the cells rather than being incorporated; Moreover, the major site of lysine binding seems to be the complexed glycoproteins IIb-IIIa.     

Role of the phospholipid binding sites,PX of p47phox and PB region of Rac1, in the formation of the phagocyte NADPH oxidase complex NOX2

In phagocytes, superoxide anion (O₂⁻), the precursor of reactive oxygen species, is produced by the NADPH oxidase complex to kill pathogens. Phagocyte NADPH oxidase consists of the transmembrane cytochrome b₅₅₈ (cyt b₅₅₈) and four cytosolic components: p40^phox, p47^phox, p67^phox, and Rac1/2. The phagocyte activation by stimuli leads to activation of signal transduction pathways. This is followed by the translocation of cytosolic components to the membrane and their association with cyt b₅₅₈ to form the active enzyme.To investigate the roles of membrane-interacting domains of the cytosolic proteins in the NADPH oxidase complex assembly and activity, we used giant unilamellar phospholipid vesicles (GUV). We also used the neutrophil-like cell line PLB-985 to investigate these roles under physiological conditions. We confirmed that the isolated proteins must be activated to bind to the membrane. We showed that their membrane binding was strengthened by the presence of the other cytosolic partners, with a key role for p47^phox. We also used a fused chimera consisting of p47^phox(aa 1–286), p67^phox(aa 1–212) and Rac1Q61L, as well as mutated versions in the p47^phox PX domain and the Rac polybasic region (PB). We showed that these two domains have a crucial role in the trimera membrane-binding and in the trimera assembly to cyt b₅₅₈. They also have an impact on O₂.- production in vitro and in cellulo: the PX domain strongly binding to GUV made of a mix of polar lipids; and the PB region strongly binding to the plasma membrane of neutrophils and resting PLB-985 cells.     

High-affinity binding ofl-glutamate to chick retinal membranes

Binding ofl-[³H]glutamate to membranes from whole chick retina and from subcellular fractions enriched with photoreceptor terminals (P₁), or terminals from the inner plexiform layer (P₂) was studied. Na⁺-dependent and Na⁺-independent binding to these membranes was demonstrated. Na⁺-independent binding was stereospecific. Kinetic analysis of the binding process indicated a single high-affinity system (K _B=0.55 M) with a capacity of approximately 20 pmoles/mg protein in all the membrane fractions. [³H]Glutamate binding to P₁ and P₂ fractions was effectively displaced by several structural analogues of glutamate. Glutamate diethyl-ester appreciably displaced binding, whereas kainic acid did not displace bound glutamate. Data indicate the binding of [³H]glutamate to physiologically relevant receptors in the chick retina.     

Ca binding to the human red cell membrane: Characterization of membrane preparations and binding sites

Summary Inside out and right side out vesicles were used to study the sidedness of Ca binding to the human red cell membrane. It was shown that these vesicles exhibited only a limited permeability to Ca, enabling the independent characterization of Ca binding to the extracellular and cytoplasmic membrane surfaces. Ca binding was studied in 10 mM Tris HCl at pH 7.4, 22±2°C and was shown to be complete in under 5 min. Scatchard plots were made from Ca binding data obtained at free Ca concentrations in the range of 10^–6 to 10^–3M. Under these conditions inside out vesicles exhibit two independent binding sites for Ca with association constants of 1×10⁵ and 6×10³ M^–1, and right side out vesicles exhibit three independent binding sites with association constants of 2×10⁵, 1.4×10⁴ and 3×10²M^–1. Upon the addition of 0.1M KCl a third, high affinity site was found on inside out vesicles with an association constant of 3×10⁵, (in 0.1 M KCl). Ca binding to inside out vesicles increased nearly linearly with pH in the, range of pH 4 to pH 11, while binding to right side out vesicles remained practically unchanged in the range of pH 7 to pH 9. Progressive increase of the ionic strength of the medium by the addition of K, Mg or Tris decreased Ca binding to inside out vesicles as did the addition of ATP. Comparison of a series of cation competitors for Ca binding sites on inside out vesicles at 0.003 mM Ca showed that La was the most effective competitor of all while Cd was the most effective divalent cation competitor of those tested. Our findings suggest that the effects of low concentrations of Ca at the inner surface of the red cell membrane are mediated primarily through Ca binding to site 1 (and, possibly site 2) of inside out vesicles of which there are approximately 1.6×10⁵ per equivalent cell.     

Bioactivation of cysteine conjugates of 1-nitropyrene oxides by cysteine conjugate beta-lyase purified from Peptostreptococcus magnus.

To determine the role of cysteine conjugate beta-lyase (beta-lyase) in the metabolism of mutagenic nitropolycyclic aromatic hydrocarbons, we determined the effect of beta-lyase on the mutagenicities and DNA binding of cysteine conjugates of 4,5-epoxy-4,5-dihydro-1-nitropyrene (1-NP 4,5-oxide) and 9,10-epoxy-9,10-dihydro-1-nitropyrene (1-NP 9,10-oxide), which are detoxified metabolites of the mutagenic compound 1-nitropyrene. We purified beta-lyase from Peptostreptococcus magnus GAI0663, since P. magnus is one of the constituents of the intestinal microflora and exhibits high levels of degrading activity with cysteine conjugates of 1-nitropyrene oxides (1-NP oxide-Cys). The activity of purified beta-lyase was optimal at pH 7.5 to 8.0, was completely inhibited by aminooxyacetic acid and hydroxylamine, and was eliminated by heating the enzyme at 55 degrees C for 5 min. The molecular weight of beta-lyase was 150,000, as determined by fast protein liquid chromatography. S-Arylcysteine conjugates were good substrates for this enzyme. As determined by the Salmonella mutagenicity test, 5 ng of beta-lyase protein increased the mutagenicity of the cysteine conjugate of 1-NP 9,10-oxide (10 nmol per plate) 4.5-fold in Salmonella typhimurium TA98 and 4.1-fold in strain TA100. However, beta-lyase had little effect on the cysteine conjugate of 1-NP 4,5-oxide (10 nmol per plate). Both conjugates exhibited only low levels of mutagenicity with nitroreductase-deficient strain TA98NR. In vitro binding of 1-NP oxide-Cys to calf thymus DNA was increased by adding purified beta-lyase or xanthine oxidase.(ABSTRACT TRUNCATED AT 250 WORDS)