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PubMed2004

Purification and characterization of extracellular Pseudomonas aeruginosa urate oxidase enzyme.

Urate oxidase (uricase) was isolated and purified from Pseudomonas aeruginosa to apparent homogeneity using ammonium sulphate precipitation followed by ion exchange and gel filtration chromatography. The specific activity of the purified uricase enzyme was found to be 636.36 with the use of uric acid as a substrate. The purified uricase enzyme is a monomeric protein with molecular weight of 64 kilodaltons. The optimal pH and temperature of the purified enzyme is 9.0 and 30 degrees C, respectively. The effect of some metal ions was studied. Sulphate forms of Fe+2, Zn+2 and Co+2 inhibit the uricolytic activity whereas; NaCl and CaCl2 enhance the enzyme activity. Moreover, the purified enzyme is inhibited by EDTA and KCN.

Pseudomonas aeruginosa↗
PubMed1979

Degradation of peroxisomal catalase and urate oxidase of rat liver.

Urate oxidase and catalase were purified from rat liver peroxisomes, and respective antibodies were prepared from rabbits by the administration of these enzymes. Although urate oxidase generally precipitates in immunoprecipitation-possible pH ranges (pH 4.5--9.5), the enzyme remained soluble in 50 mM glycine buffer (pH 9.5) containing 50% glycerol up to concentration of 0.3 mg/ml. Anti-urate oxidase reacted with purified urate oxidase as well as with the crude preparation. After [3H]leucine was injected to rats, urate oxidase and catalase were purified from rat liver at certain intervals, and further precipitated by respective antibodies. The half-life of the catalase was 39 h and that of urate oxidase, 20 h. When the sonicated light mitochondrial fraction was incubated at 37 degrees C and at pH 7.0 or 5.6, inactivation of catalase did not seem to differ between these pH values, and approximately 80% of the catalase activity remained even after 8 h. Urate oxidase was inactivated very rapidly at pH 5.6; only 30% of its activity survived incubation for 6 h. This inactivation was found to occur by some proteolytic process. From these findings, the turnover rate of urate oxidase was found to be different from that of catalase, and this distinction seemed to be due to different sensitivity to some degradative enzymes.

Animals↗
PubMed1992

Two independent mutational events in the loss of urate oxidase during hominoid evolution.

Urate oxidase was lost in hominoids during primate evolution. The mechanism and biological reason for this loss remain unknown. In an attempt to address these questions, we analyzed the sequence of urate oxidase genes from four species of hominoids: human (Homo sapiens), chimpanzee (Pan troglodytes), orangutan (Pongo pygmaeus), and gibbon (Hylobates). Two nonsense mutations at codon positions 33 and 187 and an aberrant splice site were found in the human gene. These three deleterious mutations were also identified in the chimpanzee. The nonsense mutation at codon 33 was observed in the orangutan urate oxidase gene. None of the three mutations was present in the gibbon; in contrast, a 13-bp deletion was identified that disrupted the gibbon urate oxidase reading frame. These results suggest that the loss of urate oxidase during the evolution of hominoids could be caused by two independent events after the divergence of the gibbon lineage; the nonsense mutation at codon position 33 resulted in the loss of urate oxidase activity in the human, chimpanzee, and orangutan, whereas the 13-bp deletion was responsible for the urate oxidase deficiency in the gibbon. Because the disruption of a functional gene by independent events in two different evolutionary lineages is unlikely to occur on a chance basis, our data favor the hypothesis that the loss of urate oxidase may have evolutionary advantages.

Amino Acid Sequence↗
PubMed1991

Purification and molecular properties of urate oxidase from Chlamydomonas reinhardtii.

Urate oxidase (urate: oxygen oxidoreductase, EC 1.7.3.3) from the unicellular green alga Chlamydomonas reinhardtii has been purified to electrophoretic and immunological homogeneity by a procedure which includes as main steps ammonium sulfate fractionation, gel filtration, ion exchange and xanthine-agarose affinity chromatography. The native enzyme has a relative molecular mass (Mr) of 124,000 and consists of four identical or similar-sized subunits of Mr 31,000 each. The enzyme has a Stokes's radius of 3.87 nm, a sedimentation coefficient of 6.8 S and an f/f0 of 1.23, and exhibits its maximal absorption at 276 nm. Optimum pH was 8.5 and maximum activity was shown at 40 degrees C, with an activation energy of 53 kJ.mol-1 and a Q10 of 1.96. Absorption spectrum of native reduced enzyme showed two transient maxima at 392 and 570 nm, very similar to those of metal-urate complexes, which disappeared in the presence of cyanide. Inhibition by cyanide and neocuproin, but not by salicylhydroxamic acid, strongly suggests that copper is the metal involved in enzymatic urate oxidation. By using a sensitive photokinetic method for copper determination, a content of 4 mol of copper per mol of enzyme has been found.

Calorimetry↗
PubMed1981

Properties of two urate oxidases modified by the covalent attachment of poly(ethylene glycol).

Poly(ethylene glycol) of 5 000 daltons has been attached covalently to preparations of urate oxidase (urate: oxygen oxidoreductase, EC 1.7.3.3) from hog liver and Candida utilis. Attachment of sufficient poly(ethylene glycol) to either urate oxidase renders the enzyme incapable of eliciting antibody production in mice, or of reacting with antibodies to the unmodified enzyme. The poly(ethylene glycol) : urate oxidase conjugates exhibit higher Km and lower V values than the unmodified urate oxidases. Optimal pH values are increased for the poly(ethylene glycol) : urate oxidases, and optimal temperatures are decreased. The blood circulating lives of the modified urate oxidases following intravenous injection are much longer than those of the unmodified urate oxidases: repetitive injections over a period of 90 days dd not alter the blood circulating lives of the poly(ethylene glycol) : urate oxidases. The unmodified enzymes, on the other hand, were cleared from the blood with extreme rapidity after a few intravenous injections.

Animals↗
PubMed1997

Kinetic mechanism and cofactor content of soybean root nodule urate oxidase.

The kinetic mechanism of urate oxidase isolated from soybean root nodules has been determined by initial velocity kinetic studies monitoring oxygen uptake, in order to avoid potential artifacts in the spectrophotometric assay which arise from absorbance due to unidentified products of the enzymatic reaction. Urate and O2 bind to the enzyme sequentially; xanthine is a competitive inhibitor versus urate and a noncompetitive inhibitor versus O2, which suggests that urate binds to the enzyme before O2. This kinetic mechanism was confirmed by an 18O isotope-trapping experiment, which demonstrated that O2 does not bind productively to the enzyme in the absence of urate. The pH dependence of V and (V/K)urate reveal the presence of an ionizable residue on the enzyme with a pK of approximately 6.2, which must be unprotonated for the catalytic reaction to occur. The (V/K)O2 profile is pH independent; these data are accomodated by a model in which a unimolecular step intervenes between the binding of urate and O2. The pKi profile for 9-methylurate, a competitive inhibitor versus urate, is pH independent, confirming that the protonation state of the ionizable residue is not important for binding. The pKi profile for xanthine defines a pK of 7.4, which demonstrates that the monoanion of xanthine binds to the enzyme; by analogy, the monoanion of urate is predicted to be the substrate. The four isomeric N-methylurates were examined as potential inhibitors of urate oxidase. Only 9-methylurate showed significant inhibition suggesting that ionization at N9 of urate is not required for binding; it is proposed that the N3-deprotonated urate monoanion is the species which binds to urate oxidase. The gene encoding urate oxidase was cloned from soybeans and expressed in Escherichia coli. The metal content of the recombinant enzyme was examined by inductively coupled argon plasma emission spectroscopy, and only trace quantities of copper were found. The molecular mass of the protein was determined by MALDI-TOF mass spectrometry and found to be 35,059.8 Da. The calculated molecular mass of urate oxidase is 35,052 Da; therefore, these data suggest that there is no covalently bound cofactor in urate oxidase.

Cloning, Molecular↗
PubMed1988

Tissue specificity and species differences in the distribution of urate oxidase in peroxisomes.

The localization of urate oxidase in different tissues of rat and in the livers of selected mammalian species was investigated by immunoblot analysis and protein A-gold immunoelectron microscopy. Urate oxidase was purified from rat liver and used as an antigen to generate polyclonal antibodies in the rabbit. The antibodies were found to be monospecific by immunodiffusion and immunoblot analyses. By immunoblot analysis, urate oxidase was detected in the livers of rat, two strains of mice, hamster, dog, cat, and cow, but not in the Cynomolgus monkey and human liver. Urate oxidase was not detected by immunoblot method in rat kidney, jejunal mucosa, adrenal gland, testis, and pancreas. The subcellular localization of urate oxidase was ascertained by the protein A-gold immunocytochemical staining of the Lowicryl K4M embedded tissues. Urate oxidase was localized exclusively in the crystalloid core of the peroxisome in hepatic parenchymal cells of rat, mouse, hamster, dog, cat, and cow. The limiting membrane and the matrix of hepatic peroxisomes in these species were negative for the staining. The marginal plates of feline, canine, and bovine hepatic peroxisomes were also negative for urate oxidase. This enzyme was also not detected within the peroxisomes of human and monkey livers by the immunocytochemical technique. Peroxisomes (microperoxisomes) in extrahepatic rat tissues did not stain positively for urate oxidase by the protein A-gold immunocytochemical method, although they were positive for catalase. Fatty acyl-CoA oxidase was present in peroxisomes of jejunal mucosa, Leydig cells of test-is and pancreas but not in adrenal gland. Administration of a hepatic peroxisome proliferator, ciprofibrate or Wy-14643, failed to induce urate oxidase in rat liver. These results indicate that urate oxidase is a liver specific protein in rat and its localization within the liver peroxisomes of six mammals, excluding man and a nonhuman primate, and that its localization is limited exclusively to the crystalloid core. Unlike fatty acyl-CoA oxidase, urate oxidase does not appear to be inducible significantly by peroxisome proliferator treatment in the rat liver.

Animals↗
PubMed1981

Purification and some properties of urate oxidase from nitrogen-fixing nodules of cowpea.

Urate oxidase (urate: oxygen oxidoreductase, EC 1.7.3.3) was purified 166-fold from nitrogen-fixing root nodules of cowpea Vigna unguiculata [L.] Walp. The purified enzyme showed a specific activity of 5.7 mumol urate oxidised/min per mg protein, a molecular mass of 100 kdaltons, pH optimum between 9 and 10, isoelectric point at PH 6.8, Km(urate) = 18 muM and Km(oxygen) = 29 muM. A number of metal complexing and chelating reagents were inhibitory, as were divalent cations, including Cu2+. Iron stimulated the enzyme. Low concentrations of ammonia, glutamine and xanthine were also inhibitory. The regulation of urate oxidase in relation to the assimilation of fixed nitrogen in legume nodules is discussed.

Ammonia↗
PubMed1989

Urate oxidase: primary structure and evolutionary implications.

Urate oxidase, or uricase (EC 1.7.3.3), is a peroxisomal enzyme that catalyzes the oxidation of uric acid to allantoin in most mammals. In humans and certain other primates, however, the enzyme has been lost by some unknown mechanism. To identify the molecular basis for this loss, urate oxidase cDNA clones were isolated from pig, mouse, and baboon, and their DNA sequences were determined. The mouse urate oxidase open reading frame encodes a 303-amino acid polypeptide, while the pig and baboon urate oxidase cDNAs encode a 304-amino acid polypeptide due to a single codon deletion/insertion event. The authenticity of this single additional codon was confirmed by sequencing the mouse and pig genomic copies of the gene. The urate oxidase sequence contains a domain similar to the type 2 copper binding motif found in other copper binding proteins, suggesting that the copper ion in urate oxidase is coordinated as a type 2 structure. Based upon a comparison of the NH2-terminal peptide and deduced sequences, we propose that the maturation of pig urate oxidase involves the posttranslational cleavage of a six-amino acid peptide. Two nonsense mutations were found in the human urate oxidase gene, which confirms, at the molecular level, that the urate oxidase gene in humans is nonfunctional. The sequence comparisons favor the hypothesis that the loss of urate oxidase in humans is due to a sudden mutational event rather than a progressive mutational process.

Amino Acid Sequence↗
PubMed1985

On the loss of uricolytic activity during primate evolution--I. Silencing of urate oxidase in a hominoid ancestor.

Urate oxidase activity is not detectable in liver homogenates from the gibbon, orangutan, chimpanzee, gorilla and human. Liver homogenates from five genera of Old World and two genera of New World monkeys have easily detectable levels of urate oxidase activity. There is no evidence for extant detectable intermediate steps in the loss of urate oxidase activity in the hominoids. Urate oxidase activity from Old World and New World monkeys is stable, a simple observation which debunks a long-standing myth. Urate oxidase activity was silenced in an ancestor to the five living genera of hominoids after divergence from the Old World monkeys.

Animals↗
PubMed2002

A colorimetric 96-well microtiter plate assay for the determination of urate oxidase activity and its kinetic parameters.

Urate oxidase (E.C.1.7.3.3; uricase, urate oxygen oxidoreductase) is an enzyme of the purine breakdown pathway that catalyzes the oxidation of uric acid in the presence of oxygen to allantoin and hydrogen peroxide. A 96-well plate assay measurement of urate oxidase activity based on hydrogen peroxide quantitation was developed. The 96-well plate method included two steps: an incubation step for the urate oxidase reaction followed by a step in which the urate oxidase activity is stopped in the presence of 8-azaxanthine, a competitive inhibitor. Hydrogen peroxide is quantified during the second step by a horseradish peroxidase-dependent system. Under the defined conditions, uric acid, known as a radical scavenger, did not interfere with hydrogen peroxide quantification. The general advantages of such a colorimetric assay performed in microtiter plates, compared to other methods and in particular the classical UV method performed with cuvettes, are easy handling of large amounts of samples at the same time, the possibility of automation, and the need for less material. The method has been applied to the determination of the kinetic parameters of rasburicase, a recombinant therapeutic enzyme.

Allantoin↗
PubMed1996

Cloning, sequence analysis, and expression in Escherichia coli of the gene encoding the Candida utilis urate oxidase (uricase).

A urate oxidase (uricase) gene was cloned from Candida utilis with an oligonucleotide probe based on the amino acid sequence of cyanogen bromide-cleaved uricase. The uricase gene contains 909 base pairs and encodes a protein with a predicted mass of 34,193 Da. Candida uricase was similar (49% match in amino acid sequence) to the uricase from Aspergillus flavus. The uricase from Candida utilis has four cysteines and one of them, Cys168, participates in the enzyme activity. This enzyme was expressed to a level of about 20% of total cellular protein in an Escherichia coli cell as a soluble and functional form.

Amino Acid Sequence↗
PubMed1981

Purification and properties of urate oxidase from Streptomyces cyanogenus.

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

Amino Acids↗
PubMed1998

Efficacy of urate oxidase (uricozyme) in tumour lysis induced urate nephropathy.

Urate oxidase (uricozyme) is an enzyme of non-human origin capable of oxidizing human uric acid to allantoin, a highly soluble product at renal tubule pH. We report its efficacy in three patients with acute urate nephropathy due to tumour lysis in chronic lymphatic leukaemia and high grade lymphoma. Two patients had an additional obstructive nephropathy due to ureteric urate crystals. An intravenous infusion (100 units/kg in 50 ml saline over 30 min) was given for between two and five consecutive days. All patients showed a rapid fall in serum urate levels with associated diuresis, correction of metabolic disturbance and full resolution of uraemia within a week. The treatment was well tolerated and caused a rapid resolution of clinical symptoms in all cases. We review the literature relating to the use of this agent both in the treatment of hyperuricaemic acute renal failure and gouty arthritis.

Aged↗
PubMed1995

Functional expression and peroxisomal targeting of rat urate oxidase in monkey kidney cells.

Humans and hominoid primates lack the enzyme urate oxidase, which catalyzes the oxidation of uric acid to allantoin. In rats and most other mammals, urate oxidase is present as a crystalloid core within the peroxisomes of liver parenchymal cells. To determine whether functionally active recombinantly expressed urate oxidase can be targeted to the peroxisome as well as display the crystalloid core-like structure, we expressed rat urate oxidase cDNA in African green monkey kidney cells (CV-1 cells) under the control of a cytomegalovirus promoter. Cell lines stably expressing urate oxidase were isolated. Northern blot analysis revealed a 1.3-kb transcript and immunoblot analysis confirmed the presence of urate oxidase in the stably transfected cells. The recombinant urate oxidase expressed in CV-1 cells was functionally active. Immunofluorescence microscopy revealed that the expressed protein was visualized as discrete granules in the cytoplasm. Electron microscopy and immunocytochemical localization studies showed that the recombinantly expressed protein formed distinct crystalloid core structures with bundles of tubules within single membrane limited cytoplasmic organelles. On cross section, the recombinant urate oxidase tubular structures are arranged as circles of 10 surrounding a slightly larger circle. This arrangement is reminiscent of urate oxidase-containing cores in rat liver peroxisomes. Immunocytochemical studies confirmed that the recombinantly expressed urate oxidase is correctly targeted to the catalase-containing peroxisomes in these CV-1 cells.

Allantoin↗
PubMed1982

Ultrastructural localization of urate oxidase in nodules of Sesbania exaltata, Glycine max, and Medicago sativa.

The localization of urate oxidase (=uricase, E.C. 1.7.3.3) was determined cytochemically in nodules of Sesbania exaltata (Raf.) Cory, soybean (Glycine max [L.] Merr.) and alfalfa (Medicago sativa [L.] ), using the precipitation of peroxide (produced during the oxidation of urate) by cerium chloride. Cerium perhydroxide reaction product was noted only in the microbodies, a localization consistent with biochemical fractionation studies on urate oxidase. Urate oxidase was present not only in the uninfected cells of the cortical tissue, but also in both infected and interstitial cells in the central tissue, suggesting that at least this enzyme of ureide metabolism is not confined to interstitial cells. Urate oxidase cytochemistry of nodules from alfalfa (Medicago sativa L.), an amide producer, also resulted in microbody staining but the microbodies were infrequently noted in cell profiles.

Fabaceae↗
PubMed1992

Rat urate oxidase produced by recombinant baculovirus expression: formation of peroxisome crystalloid core-like structures.

Urate oxidase (EC 1.7.3.3), which catalyzes the oxidation of uric acid to allantoin, is present in most mammals but absent in humans and hominoid primates. In rats and most other mammals that catabolize uric acid to allantoin, this enzyme is localized within the crystalloid cores of peroxisomes present in liver parenchymal cells. To determine whether urate oxidase forms these crystalloid cores or whether core-forming protein(s) exist in association with urate oxidase, a baculovirus expression vector system was used to overproduce the full-length rat urate oxidase in Spodoptera frugiperda cells. Urate oxidase was expressed to a level of approximately 30% of the total protein in this system. Immunoblot analysis demonstrated that the baculovirus-generated protein had electrophoretic and immunologic properties similar to those of urate oxidase expressed in rat liver. Immunofluorescence and electron microscopic examination revealed that the overexpressed recombinant urate oxidase is present in both the cytoplasm and the nucleus of infected insect cells as numerous 1- to 3-microns discrete particles. These insoluble protein aggregates, which were positively stained for urate oxidase by protein A-gold immunocytochemical approach, did not appear to be delimited by a single membrane. They revealed a crystalloid structure reminiscent of rat peroxisomal core consisting of bundles of tubules with an inner diameter of approximately 50 A. The recombinant urate oxidase particles, isolated by a single-step procedure, were composed entirely of 35-kDa urate oxidase subunit. These studies indicate that rat urate oxidase is capable of forming insoluble crystalloid core-like structures.

Animals↗
PubMed1978

[Experimental study of the immune response to swine liver urate oxidase].

An immune response to urate oxidase--an enzyme from pig liver was studied. Peculiarities of antibody formation were investigated in three animal species. It was shown that the scheme for repeated injections of the enzyme preparation had a marked effect on the intensity of humoral immune response. In studying the process of the enzyme allergic properties it was found that urate oxidase had anaphylactogenic and skin sensitizing properties. The strength of anaphylactic reaction depened on the sensitizing dose of the antigen and correlated with the results of active skin anaphylaxis. However, there was no correlation between the strength of the immune response determined by means of the indirect hemagglutination test and the intensity of anaphylactic reaction.

Allergens↗