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PubMed1983

On the synthesis and incorporation of catalase and urate oxidase into the peroxisomes of mouse liver.

The processes associated with the biogenesis of peroxisomes in mouse liver have been studied by following the incorporation of radiolabelled leucine into major enzymic components of this organelle. Maximal incorporation of label into peroxisomal catalase and urate oxidase occurred within 2 hr, with the urate oxidase being labelled before catalase, but subsequent to the incorporation of phospholipid into this organelle. Subsequently, immunoprecipitation of catalase from the large granular fraction of mouse liver was shown to result in the isolation of a catalase molecule which had lost a peptide of approx. 2000 dalton from each subunit by comparison with the newly-synthesized enzyme. It was observed that the modification of catalase was obviated by the presence of leupeptin and iodoacetamide and this information has enabled the purification of both modified and unmodified forms of the enzyme. The possible significance of these data has been discussed and the major features incorporated into a working model of peroxisomal biogenesis.

Animals↗
PubMed1965

Combined biochemical and morphological study of particulate fractions from rat liver. Analysis of preparations enriched in lysosomes or in particles containing urate oxidase, D-amino acid oxidase, and catalase.

Six particulate preparations isolated from rat liver under different experimental conditions were analyzed biochemically and examined in the electron microscope. The results confirm the lysosomal nature of the pericanalicular dense bodies and demonstrate that the microbodies are the bearers of urate oxidase, catalase, and D-amino acid oxidase. Catalase, representing a major component of the particles, and D-amino acid oxidase appear to be associated with the structureless "sap" of the particles, urate oxidase with their crystalloid core or with their outer membrane.

Animals↗
PubMed2004

Complexed and ligand-free high-resolution structures of urate oxidase (Uox) from Aspergillus flavus: a reassignment of the active-site binding mode.

High-resolution X-ray structures of the complexes of Aspergillus flavus urate oxidase (Uox) with three inhibitors, 8-azaxanthin (AZA), 9-methyl uric acid (MUA) and oxonic acid (OXC), were determined in an orthorhombic space group (I222). In addition, the ligand-free enzyme was also crystallized in a monoclinic form (P2(1)) and its structure determined. Higher accuracy in the three new enzyme-inhibitor complex structures (Uox-AZA, Uox-MUA and Uox-OXC) with respect to the previously determined structure of Uox-AZA (PDB code 1uox) leads to a reversed position of the inhibitor in the active site of the enzyme. The corrected anchoring of the substrate (uric acid) allows an improvement in the understanding of the enzymatic mechanism of urate oxidase.

Amino Acid Sequence↗
PubMed2005

Recombinant urate oxidase (rasburicase) in the prophylaxis and treatment of tumor lysis syndrome.

Spontaneous or treatment-induced tumor lysis syndrome (TLS) can cause significant morbidity and potential mortality. Vigorous hydration, alkalinization and inhibition of uric acid synthesis are the most frequently used methods for prevention of TLS. However, this approach requires hospitalization and tedious nursing care, and fails to prevent renal insufficiency in up to 25% of high-risk patients. With increased intensity and efficacy of cancer therapies, and the current trend to deliver treatment in the outpatient setting, novel approaches at management of TLS are needed. Unlike allopurinol, urate oxidase promptly reduces the existing uric acid pool, prevents accumulation of xanthine and hypoxanthine, and does not require alkalinization, facilitating phosphorus excretion. A recombinant form of urate oxidase, rasburicase, is now available. In this chapter we will present an overview of rasburicase development and discuss the impact of rasburicase in the prevention and management of TLS.

Allopurinol↗
PubMed1992

Cloning and expression in Escherichia coli of the gene encoding Aspergillus flavus urate oxidase.

Amino acid sequencing of peptides obtained after proteolytic hydrolysis of Aspergillus flavus urate oxidase (uricase) permitted the design of oligodeoxynucleotide probes that were used to obtain 1.2- and 5-kilobase pair DNA fragments from A. flavus cDNA and genomic libraries, respectively. The cDNA fragment contained the entire coding region for uricase, and comparison with the genomic fragment revealed the presence of two short introns in the coding region of the gene. A. flavus uricase has around 40% overall identity with uricases from higher organisms but with many conserved amino acids. Hitherto highly conserved consensus patterns found in other uricases were found to be modified in the A. flavus enzyme, notably the sequence Val-Leu-Lys-Thr-Thr-Gln-Ser near position 150, which in the filamentous fungus is uniquely modified to Val-Leu-Lys-Ser-Thr-Asn-Ser. Silent mutations were introduced by cassette mutagenesis near the 5'-extremity of the coding sequence in order to conform with Escherichia coli codon usage, and the uricase was expressed in the E. coli cytoplasm in a completely soluble, biologically active form.

Amino Acid Sequence↗
PubMed1990

Molecular characterization of the Drosophila melanogaster urate oxidase gene, an ecdysone-repressible gene expressed only in the malpighian tubules.

The urate oxidase (UO) gene of Drosophila melanogaster is expressed during the third-instar larval and adult stages, exclusively within a subset of cells of the Malpighian tubules. The UO gene contains a 69-base-pair intron and encodes mature mRNAs of 1,224, 1,227, and 1,244 nucleotides, depending on the site of 3' endonucleolytic cleavage prior to polyadenylation. A direct repeat, 5'-AAGTGAGAGTGAT-3', is the proposed cis-regulatory element involved in 20-hydroxyecdysone repression of the UO gene. The deduced amino acid sequences of UO of D. melanogaster, rat, mouse, and pig and uricase II of soybean show 32 to 38% identity, with 22% of amino acid residues identical in all species. With use of P-element-mediated germ line transformation, 826 base pairs 5' and approximately 1,200 base pairs 3' of the D. melanogaster UO transcribed region contain all of the cis elements allowing for appropriate temporal regulation and Malpighian tubule-specific expression of the UO gene.

Amino Acid Sequence↗
PubMed1991

Species differences in the temporal pattern of Drosophila urate oxidase gene expression are attributed to trans-acting regulatory changes.

The Drosophila melanogaster urate oxidase (UO)-encoding gene is expressed in the third-instar larva and adult. In contrast, the Drosophila pseudoobscura UO gene is only expressed in the adult, whereas the Drosophila virilis UO gene is expressed only in the third-instar larva. UO activity in these three Drosophila species is detected exclusively within the Malpighian tubules. By using P-element mediated germ-line transformation, UO genes from D. pseudoobscura and D. virilis were integrated into the D. melanogaster genome. The D. virilis and D. pseudoobscura UO transgenes were expressed in the third-instar larva and adult Malpighian tubules, which is the D. melanogaster temporal pattern of UO gene expression. These observations indicate that differences in the temporal patterns of regulation of UO genes among these three Drosophila species are not likely to be due to evolutionary changes in the sequence or complement of UO cis-acting regulatory elements. The species differences in UO regulation are probably the result of changes in one or more trans-acting factors required for UO gene expression in the third-instar larval and adult stages.

Age Factors↗
PubMed1992

The urate oxidase gene of Drosophila pseudoobscura and Drosophila melanogaster: evolutionary changes of sequence and regulation.

The urate oxidase (UO) transcription unit of Drosophila pseudoobscura was cloned, sequenced, and compared to the UO transcription unit from Drosophila melanogaster. In both species the UO coding region is divided into two exons of approximately equal size. The deduced D. pseudoobscura and D. melanogaster UO peptides have 346 and 352 amino acid residues, respectively. The nucleotide sequences of the D. pseudoobscura and D. melanogaster UO protein-coding regions are 82.2% identical whereas the deduced amino acid sequences are 87.6% identical with 42 amino acid changes, 33 of which occur in the first exon. Although the UO gene is expressed exclusively within the cells of the Malpighian tubules in both of these species, the temporal patterns of UO gene activity during development are markedly different. UO enzyme activity, UO protein, and UO mRNA are found in the third instar larva and adult of D. melanogaster but only in the adult stage of D. pseudoobscura. The intronic sequences and the extragenic 5' and 3' flanking regions of the D. pseudoobscura and D. melanogaster UO genes are highly divergent with the exception of eight small islands of conserved sequence along 772 bp 5' of the UO protein-coding region. These islands of conserved sequence are possible UO cis-acting regulatory elements as they reside along the 5' flanking DNA of the D. melanogaster UO gene that is capable of conferring a wild-type D. melanogaster pattern of UO regulation on a UO-lacZ fusion gene.

Amino Acid Sequence↗
PubMed2003

Altered antigenic disposition of peroxisomal urate oxidase in PEX5-defective Chinese hamster ovary cells.

Since Chinese hamster ovary (CHO) cells never express urate oxidase (UO), we tried to establish cell lines stably producing UO in order to elucidate the peroxisomal import process. The enzyme is a peroxisome targeting signal 1 (PTS1) protein harboring SKL motif at the carboxy-terminus [Biochem. Biophys. Res. Commun. 158 (1989) 991] and PEX5 protein (Pex5p) is supposed to be involved in the import process [Nat. Genet. 9 (1995) 115; J. Cell Biol. 130 (1995) 51]. We transfected a cDNA encoding rat UO into both wild type and PEX5-defective CHO cells to isolate each cell line stably producing the enzyme. While we examined the import process of UO in mutant cells, we noticed an interesting observation by using polyclonal antibody U1 or U2, which separately recognizes epitopes of UO. U1 antibody mainly interacts with epitopes in the amino-terminal region of UO. On the other hand, U2 antibody reacts with many epitopes distributed in the broad region of UO molecule. When UO produced in cultured cells was stained with U2 antibody, the enzyme was detected in peroxisomes of both wild type and PEX5-mutant cells. Whereas, U1 antibody stained the peroxisomal UO in wild type cells, but not in PEX5-mutant cells. These immunocytochemical observations suggest that the epitopes at the amino-terminal region of UO will be concealed in mutant cells. When the mutant cells were transfected with wild type PEX5 cDNA, U1 antibody came to react with UO in peroxisomes of mutant cells. The restoration indicates that the exposure of N-terminal epitopes of UO will depend upon the functional Pex5p. Immunoelectron microscopic observation showed that the peroxisomal import of UO was partially retarded in PEX5 mutant cells. The observation also supported the fact that UO was mainly localized in the peroxisomal matrix of wild type cells but in the membrane of mutant cells.

Animals↗
PubMed1996

Transformation of epithelial cells stably transfected with H2O2-generating peroxisomal urate oxidase.

Peroxisome proliferators, a group of structurally diverse nongenotoxic agents, induce predictable pleiotropic responses in liver, including the development of liver tumors in rats and mice. These agents transcriptionally activate the three genes of the peroxisomal beta oxidation enzyme system by interacting with the peroxisome proliferator-activated receptor(s). It has been proposed that H2O2 generated by the peroxisomal beta oxidation system leads to DNA damage and neoplastic transformation. Consistent with this hypothesis is that cells stably transfected with H2O2-generating peroxisomal fatty acyl-CoA oxidase cDNA, which encodes the first and rate-limiting enzyme of the beta oxidation system, undergo transformation in the presence of a fatty acid substrate. To test whether H2O2 generated by other peroxisomal oxidases can also lead to transformation, a full-length cDNA encoding rat urate oxidase (UOX), which oxidizes uric acid to allantoin and in the process generates H2O2, was introduced into African green monkey kidney cells (CV-1 cells) under the control of constitutively active human peroxisomal fatty acyl-CoA oxidase gene promoter. Five stably transfected CV-1 cell lines expressing recombinant rat UOX were isolated in which the recombinant protein was targeted to peroxisomes and formed crystalloid structures or cores similar to those present in rat liver peroxisomes. Increased levels of H2O2 were found when cells stably expressing UOX were exposed to the substrate uric acid. These five clones, designated A-U1 to A-U5, exhibited anchorage-independent growth, as demonstrated by the formation of transformed colonies in soft agar in proportion to the duration of exposure to uric acid. These transformants exhibited clonal growth under serum-deprived conditions. One of these transformed cell lines, the A-U3 cell line, was evaluated for tumorigenicity by s.c. injection in nude mice. All five mice injected with transformed A-U3 cells developed adenocarcinomas, but no tumors developed in mice injected with control CV-1 cells or cells stably expressing UOX that were not exposed to uric acid. These results provide further evidence indicating that sustained overexpression of a peroxisomal H2O2-generating oxidase causes cell transformation.

Animals↗
PubMed1991

Rat urate oxidase: cloning and structural analysis of the gene and 5'-flanking region.

The structural gene (UOX) encoding rat urate oxidase (UOX) spans at least 23 kb and is composed of eight exons and seven introns. All of the exon-intron splice junction sequences conformed to the GT/AG consensus established for eukaryotic genes. The transcription start point (tsp) was determined using S1-type nuclease protection riboprobe, and assigned to an adenine 54 nucleotides (nt) upstream of the ATG start codon. A 456-bp 5'-terminal fragment, starting at the ATG codon, carries a putative TATA (ATAAAA) sequence at -32, and two putative 'CAAT box' sequences at -62 and -71 bp upstream from the tsp. No sequence resembling 'GC' box hexanucleotides (GGGCGG or CCGCCC) was found. The structural features of the 5'-flanking region of the UOX gene are distinct from the 5'-flanking sequences of peroxisomal beta-oxidation system genes which contain one or more 'GC' box elements but lack TATA- and CAAT-like features [Osumi et al., J. Biol. Chem. 262 (1987) 8138-8143; Ishii et al., J. Biol. Chem. 262 (1987) 8144-8150]. The 5'-flanking region of the UOX gene reveals a sequence, TTAGTAATT at nt -276 from the tsp, which appears to be complementary to the underlined part of the liver-specific LF-B1/HNF-1 consensus sequence, GTTAATNATTAAC (where N = A, C, T, G or no nt).

Amino Acid Sequence↗
PubMed2005

Urate oxidase from Aspergillus flavus: new crystal-packing contacts in relation to the content of the active site.

Urate oxidase from Aspergillus flavus (uricase or Uox; EC 1.7.3.3) is a 135 kDa homotetramer with a subunit consisting of 301 amino acids. It catalyses the first step of the degradation of uric acid into allantoin. The structure of the extracted enzyme complexed with a purine-type inhibitor (8-azaxanthin) had been solved from high-resolution X-ray diffraction of I222 crystals. Expression of the recombinant enzyme in Saccharomyces cerevisiae followed by a new purification procedure allowed the crystallization of both unliganded and liganded enzymes utilizing the same conditions but in various crystal forms. Here, four different crystal forms of Uox are analyzed. The diversity of the Uox crystal forms appears to depend strongly on the chemicals used as inhibitors. In the presence of uracil and 5,6-diaminouracil crystals usually belong to the trigonal space group P3(1)21, the asymmetric unit (AU) of which contains one tetramer of Uox (four subunits). Chemical oxidation of 5,6-diaminouracil within the protein may occur, leading to the canonical (I222) packing with one subunit per AU. Coexistence of two crystal forms, P2(1) with two tetramers per AU and I222, was found in the same crystallization drop containing another inhibitor, guanine. Finally, a fourth form, P2(1)2(1)2 with one tetramer per AU, resulted fortuitously in the presence of cymelarsan, an additive. Of all the reported forms, the I222 crystal forms present by far the best X-ray diffraction resolution (approximately 1.6 angstroms resolution compared with 2.3-3.2 angstroms for the other forms). The various structures and contacts in all crystalline lattices are compared. The backbones are essentially conserved except for the region near the active site. Its location at the dimer interface is thus likely to be at the origin of the crystal contact changes as a response to the various bound inhibitors.

Aspergillus flavus↗
PubMed1993

Characterisation, cloning and integrative properties of the gene encoding urate oxidase in Aspergillus nidulans.

A number of mutations have been obtained which define the structural gene (uaZ) coding for urate oxidase in linkage group I of Aspergillus nidulans. This gene has been cloned by transformation of a uaZ- null mutant. A chromosome I/VIII translocation which splits the gene has been defined both genetically and physically. All known mutations are contained in a 1-kb fragment, itself contained in the probe which recognizes a 1.2-kb inducible message. Plasmids carrying uaZ show a strict bias towards homologous recombination in transformation experiments.

Alleles↗
PubMed1986

Cloning a cDNA for Drosophila melanogaster urate oxidase.

A cDNA library from third-instar larval Malpighian tubules of Drosophila melanogaster was constructed and screened for urate oxidase (UO) clones by hybridization selection. The coding sequence for UO was mapped by in situ hybridization to position 28C on the left arm of chromosome 2. The UO activity in Drosophila shows a complex developmental profile. A UO cDNA was used as a probe of Northern blots of poly(A) + RNA from various stages of development. The data show that there is a direct correlation between the transcriptional activity of the UO locus as evidenced by the quantitative changes of UO mRNA and the levels of UO activity and protein during development.

Animals↗
PubMed2005

Characterization of the degradation of recombinant rat urate oxidase in tetracycline controlled gene expression cells.

Our previous study has shown that uric acid is metabolized by recombinant rat urate oxidase (UOX) and generates hydrogen peroxide leading to DNA damage and cell transformation. However, in transformed cells protein levels of UOX were reduced compared with the control cell. To investigate the characterization and the mechanisms responsible for the degradation of UOX, a controllable gene expression system has been used to switch on/off controlled expression of rat UOX in vitro. Chinese hamster ovary cells were double transfected with regulatory and responsive plasmids, pCMV-tTA and pTRE-rUOX, respectively. The cells expressing rat UOX were subtly controlled by tetracycline (Tc). High levels of UOX mRNA and protein enzymatic activity were observed when the cells were cultured in the absence of Tc. The functional recombinant rat UOX was present in the form of crystalloid cores structures that localized within the peroxisomes of the cells, which was confirmed by transmission and immunoelectron microscopic studies. The addition of Tc into the medium led to the halting of rat UOX gene transcription. As a result, recombinant rat UOX mRNA was lost rapidly followed by loss of crystalloid cores structures and UOX protein degradation. Lysosomes assembled around the UOX specific structures indicating that they were involved in degradation of the protein. The observations suggest that the entire organelle rather than a single protein within the peroxisomes is degraded once the rat UOX gene expression is turned off, and the phagocytic vacuole/lysosome pathway (microautophagic process) may play an important role in degradation of the protein under the present situation.

Animals↗
PubMed2002

Loss of urate oxidase activity in hominoids and its evolutionary implications.

We have determined and compared the promoter, coding, and intronic sequences of the urate oxidase (Uox) gene of various primate species. Although we confirm the previous observation that the inactivation of the gene in the clade of the human and the great apes results from a single CGA to TGA nonsense mutation in exon 2, we find that the inactivation in the gibbon lineage results from an independent nonsense mutation at a different CGA codon in exon 2 or from either one-base deletion in exon 3 or one-base insertion in exon 5, contrary to the previous claim that the cause is a 13-bp deletion in exon 2. We also find that compared with other organisms, the primate functional Uox gene is exceptional in terms of usage of CGA codons which are prone to TGA nonsense mutations. Nevertheless, we demonstrate rather strong selective constraint against nonsynonymous sites of the functional Uox gene and argue that this observation is consistent with the fact that the Uox gene is unique in the genome and evolutionarily conserved not only among animals but also among eukaryotes. Another finding that there are a few substitutions in the cis-acting element or CAAT-box (or both) of primate functional Uox genes may explain the lowered transcriptional activity. We suggest that although the inactivation of the hominoid Uox gene was caused by independent nonsense or frameshift mutations, the gene has taken a two-step deterioration process, first in the promoter and second in the coding region during primate evolution. It is also argued that the high concentration of uric acid in the blood of humans and nonhuman primates has developed molecular coevolution with the xanthine oxidoreductase in purine metabolism. However, it remains to be answered whether loss of Uox activity in hominoids is related to protection from oxidative damage and the prolonged life span.

Animals↗
PubMed1996

Mapping of the porcine urate oxidase and transforming growth factor beta 2 genes by fluorescence in situ hybridization.

We have mapped two genes from human chromosome 1, urate oxidase (UOX) and transforming growth factor beta 2 (TGFB2), by fluorescence in situ hybridization (FISH) in the pig genome. Porcine-specific polymerase chain reaction (PCR) primers for both genes were designed from the porcine cDNA sequence. With the help of these primers yeast artificial chromosome (YAC) clones for UOX and TGFB2 were isolated from a pig YAC library. These DNA probes were used for FISH analysis. TGFB2 was localized to SSC 10p16. With the YAC probe for UOX two porcine chromosome regions 6q26 and 6q32, revealed specific signals. These results, help to refine the comparative mapping data between human and pig.

Animals↗
PubMed1989

The nucleotide sequence of a full length cDNA clone encoding rat liver urate oxidase.

Recently we reported the sequence of a cDNA clone (pUOX-1), isolated from a lambda gt11 cDNA library, which encoded for rat liver urate oxidase (EC 1.7.3.3), but this clone lacked the nucleotide sequences encoding the N-terminal region for this enzyme. Using the cDNA insert from the pUOX-1 clone as a probe, we have now isolated a full length cDNA clone, pUOX-2, from a lambda gt10 library by plaque hybridization. Nucleotide sequence analysis of the pUOX-2 clone showed that it has 1379 base pairs with an open reading frame coding for 303 amino acid residues corresponding to a molecular mass of 34,931 daltons. In addition to the open reading frame the pUOX-2 contains 439 bp of 3'-untranslated and 41 bp of 5'-untranslated sequences. The consensus polyadenylation signal AATAAA precedes a stretch of poly(A)+ residues at the 3' end.

Amino Acid Sequence↗