Uricase Activity Research Articles

Identification of an amino acid residue involved in the substrate-binding site of rat liver uricase by site-directed mutagenesis

Computer analysis has shown that a conserved amino acid sequence (Leu 160 to Lys 164) of rat liver uricase is also present in other enzymes with purine substrates. The significances of the amino acids in this sequence were studied by site-directed mutagenesis. Replacement of Lys 164 by Glu or Ile resulted in loss of uricase activity and decrease in binding of the competitive inhibitor xanthine. The far ultraviolet circular dichroic spectra of the mutant uricases were identical to that of the wild type protein, indicating that the replacement of Lys 164 by other amino acids did not result in serious modification of the conformation of uricase. These findings suggest that this amino acid is involved in the substrate-binding site of the enzyme.

Physiological and Biochemical Aspects of Symbiotic Nitrogen Fixation in Cowpea (Vigna unguiculata(L.) Walp. var. Tuy) Plants Infected by Cowpea Mosaic Virus

Three-day-old cowpea seedlings were inoculated with the severe strain of Cowpea Mosaic Virus (CpMV) and 24 h later with Bradyrhizobium sp. cowpea, strain 1-125, when virus translocation to roots still had not taken place. Plants were harvested at 22, 30, 45 and 59 d after germination. Active virus replication was associated with increased protein content, detected in the leaves of 22-d-old plants. CpMV infection reduced the total leaf area, dry weight of shoots and the chlorophyll content of the first trifoliolate leaf at all experimental times. Low sugar content was recorded in leaves of 22- and 30-d-old CpMV-infected plants and in nodules and roots of 30- and 45-d-old CpMV-infected plants. Up to 45 d, the nodule mass of CpMV-infected plants was lower than in controls, but reached control values at the 4th harvest. In CpMV-infected nodules, massive agglomeration of virus particles, crystalline virus inclusions and the proliferation of the endoplasmic reticulum were observed only in those cells containing bacteroids. In 30- and 45-d-old plants, CpMV infection decreased the content of ureides in nodules, roots, and petioles. Virus infection did not alter the a-amino-N content of roots and nodules but induced a transient 74% reduction in the level of a-amino-N in petioles of 45-d-old plants. At the 1st and 2nd harvests the activity of uricase (EC 1.7.3.3) in the nodules and of pyruvate kinase (EC 2.7.1.40) in the nodules and leaves were decreased severely by virus infection. CpMV did not hinder the allantoinase (EC 3.5.2.5) activity in the leaves but caused a 9% transitory decrease in the activity of this enzyme in nodules of 45-d-old plants. Measurements of NAD-malic enzyme (EC 1.1.1.38) in nodules also showed the non-effect of CpMV on this enzyme, except for a temporary 16% reduction at the 2nd harvest. As compared to controls, the relative abundance of ureides in 30-d-old CpMV-infected plants indicated a 15%, 10%, and 51% reduction in the nodules, roots, and petioles, respectively. Results indicate that at the time of the 4th harvest the symbiotic process, measured in terms of ureide content and enzymatic activities, was functioning at a near normal level despite nodule infection by CpMV.

Translocation of nitrogen and expression of nodule‐specific uricase (nodulin‐35) in Robinia pseudoacacia

Uricase (urate oxidase, EC 1.7.3.3) activity and nodule‐specific uricase II (nodulin‐35) were detected in the nodules from a number of legume: Rhizobium symbioses (Vigna unguiculata (L.) Walp., Phaseolus vulgaris L., and Kennedia coccinea Vent.) in the Phaseoleae, as well as in those of Robinia pseudoacacia L. which belongs to the tribe Robineae. Neither uricase activity nor nodulin‐35 was detected in nodules from Lupinus angustifolius L., an amide‐forming symbiosis of the tribe Genisteae. Nodules of R. pseudoacacia also showed high levels of allantoinase (EC 3.5.2.5) activity but activity of enzymes earlier in the pathway of ureide synthesis (xanthine dehydrogenase, EC 1.2.1.37; inosine monophosphate dehydrogenase, EC 1.2.1.14; and xanthosine nucleosidase, EC 3.2.2.1) could not be detected. Analysis of transport fluids (xylem, phloem and nodule exudates) from R. pseudoacacia found that asparagine, and, to a lesser extent, glutamine were the major translocated nitrogenous solutes. Ureides accounted for, at most, 2.6% of the N in transport fluids (tracheal xylem sap) and in nodule exudate, 0.1%. In common with nodules of the ureide‐forming symbioses, those of R. pseudoacacia contained a high proportion of uninfected interstitial cells (53.7 ± 2.3%) in the central N2‐fixing tissue whereas in L. angustifolius only 2.5 ± 0.4% of cells in this tissue were uninfected. These data have been interpreted to indicate that expression of nodule‐specific uricase is related to the differentiation of uninfected interstitial cells in nodules and not to the synthesis of ureides.

Activities of Uricase, Xanthine Oxidase, and Xanthine Dehydrogenase in the Hepatopancreas of Aquatic and Terrestrial Crabs

Activities of Uricase, Xanthine Oxidase, and Xanthine Dehydrogenase in the Hepatopancreas of Aquatic and Terrestrial Crabs

Effect of rust infection on levels of uricase, allantoinase and ureides in susceptible and hypersensitive bean leaves

The enzymatic activities involved in two sequential steps of purine catabolism and ureide biosynthesis, i.e. uricase, catalase and allantoinase, were determined in susceptible and hypersensitive bean leaf tissue after infection with Uromyces phaseoli. Uricase increased to approx. two times more than the control value 20–22 h after inoculation of the hypersensitive cv. K.W. 765 (reaction grade 2 ++) close to pathogen penetration and first haustoria formation and, 1 day later, in the susceptible cvs Golden Gate Wax, Pinto 111 (reaction grade 6) and in the hypersensitive cv. K.W. 814 (reaction grade 1). Afterwards, the highest increases in uricase activity (up to 10 times the control), mainly localized in fractions of leaf homogenate sedimenting at 2000 g and 15000 g , were detected in c.v. K.W. 765 during lesion development, indicating that a peroxisome-associated enzyme was involved. Lower increases (up to two to three times the control) were observed in the susceptible cultivars during stroma differentiation (flecks) and uredospore formation. Catalase linked to the particulate fractions increased in susceptible cultivars to the same extent as uricase from 2 to 6 days after inoculation. Also in cv. K.W. 765 it increased during lesion development, but very much less than uricase, with the consequence that a possible toxic action, due to unscavenged H 2O 2 production, could arise during hypersensitivity expression. An enhancement of allantoinase two or three times the control values during stroma differentiation was detected in susceptible cultivars. By contrast, allantoinase activity was unchanged during local lesion formation in cv. K.W. 765, indicating that the activation of this enzyme is specifically involved during the establishment of a biotrophic relationship between Phaseolus vulgaris and Uromyces phaseoli. In the susceptible cultivar, an increase in allantoic acid during the late stages of infection (uredospores differentiation) was observed. Both allantoin and allantoic acid were enhanced during lesion development in the hypersensitive cultivar. The results are consistent with the interpretation that the enhanced purine catabolism in rust-infected bean leaves is connected both to a nutritional benefit for the parasite in the compatible relationship, and to a toxic effect for the host in the incompatible one.

Effect of high dietary concentrations of brewer's dried yeast on growth performance and liver uricase in rainbow trout ( Oncorhynchus mykiss)

The potential of using yeast nitrogen in fish feeds to replace proteins from conventional sources is an attractive option to nutritionists, feed manufacturers and fish farmers. Experiments were conducted to determine the effects of high dietary levels of brewer's dried yeast, with 20% of its protein represented by nucleic acid nitrogen, on the growth, feed conversion efficiency and uric acid metabolism of rainbow trout ( Oncorhynchus mykiss). Growth and liver uricase activities were monitored in trout fed diets containing 0, 25, 50 or 75% yeast. Growth was faster and feed conversion more efficient in fish fed diets consisting of 25% yeast; the diets containing 50 and 75% yeast were seemingly unpalatable to trout. Liver uricase activity, present in all fish, was directly correlated with the dietary level of nucleic acids. It was concluded that although feed intake was depressed at yeast levels greater than 25%, the fish did not appear to be adversely affected physiologically by high dietary levels of nucleic acid nitrogen.

Soybean Nodule-Specific Uricase (Nodulin-35) Is Expressed and Assembled into a Functional Tetrameric Holoenzyme in Escherichia coli

A complete nodulin-35 (N-35) cDNA encoding nodule-specific uricase (EC 1.7.3.3) was isolated from a soybean (Glycine max L. var. Prize) nodule cDNA expression library using a previously isolated partial cDNA clone. The N-35 cDNA was expressed in Escherichia coli driven by the lacZ promoter and was found to be functionally active. The uricase activity was detected in the cytoplasmic fraction of E. coli with the same pH optimum and apparent K(m) values as that in the nodules. Because a stop codon is located 15 base pairs upstream of the N-35 initiation codon, it appears that a fusion protein with LacZ was not made, but reinitiation occurred due to the presence of a putative Shine-Dalgarno sequence in the appropriate region. The size of the N-35 polypeptide made in E. coli is identical to that present in soybean nodules and is able to assemble into a tetrameric holoenzyme with the same molecular weight as the native uricase. Thus, the presence of peroxisomes does not appear to be essential for the proper assembly of the holoenzyme in E. coli. These data also indicate that posttranslational modifications or membrane transport are not essential either for the assembly of N-35 into a holoenzyme or for the activity of uricase.

Purineolytic capacity and origin of hemolymph urate in Carcinus maenas during hypoxia

1. 1. Purineolytic capacity has been examined in Carcinus maenas as a potential source of the urate which accumulates in hemolymph during hypoxia and which allosterically increases hemocyanin O 2 affinity. 2. 2. Hepatopancreatic xanthine dehydrogenase (XDH) activities can account for all hemolymph urate accumulating during hypoxia. However, uricase activities consistently exceed XDH, raising the question of how urate accumulates. 3. 3. Uricase is neither permanently impared by hypoxia, nor inhibited by excess substrate. Rather, uricase has a low affinity for O 2, a required cofactor ( K m = 65 mm Hg, ± 8 SD); diminished O 2 availability during hypoxia correspondingly impedes uricase activity thereby allowing urate to accumulate. 4. 4. Variability in C. maenas hemolymph urate levels (Lallier et al., 1987, Comp. Biochem. Physiol. 86A, 255–260; Lallier and Truchot, 1990, Resp. Physiol., 77, 323–336) is mirrored by similar variability in XDH and uricase activities. 5. 5. Dietary purines can easily account for urate accumulation, suggesting that adenylate catabolism is not normally a major source of hemolymph urate.

Enhanced uricase activity in tobacco mosaic virus infected tobacco leaves

The activity of uricase (urate:oxygen oxidoreductase, E.C 1.7.3.3), an enzyme implicated in oxidative purine catabolism, was determined in tobacco leaves susceptible and hypersensitive to tobacco mosaic virus (TMV) infection. An average uricase activity of 0.5–0.6 nmol min −1g −1 fresh weight and 0.2 nmol min −1 uric acid oxidized mg −1 protein was recorded in healthy tobacco total leaf extracts, while allantoinase activity and ureides were not found. Enhanced uricase activity was detected both in the compatible interaction from the 1st to 3rd post-inoculation days (infected/healthy ratio = 1.5), and in the incompatible interaction from the 2nd to 7th post-inoculation days, during local lesion differentiation (the infected/healthy value being 2.6 and 3.2 at the 4th and 7th post-inoculation day). Two considerations emerge from these results: (i) enhanced uricase activity seems to correlate with both the stimulated metabolism induced by virus replication and the necrobiotic process associated with hypersensitivity; (ii) the marked increase in uricase, H 2O 2-producing enzyme, during the hypersensitive response suggests that a consistent increase in hydrogen peroxide relase take place during development of local lesion that may be, at least in part, associated with host cell damage.

ACTIVITIES OF URICASE, XANTHINE OXIDASE, AND XANTHINE DEHYDROGENASE IN THE HEPATOPANCREAS OF AQUATIC AND TERRESTRIAL CRABS

ABSTRACT To examine the effect of habitat on urate metabolism in crabs, we measured the activities of xanthine oxidase (XOD), xanthine dehydrogenase (XDH), and uricase in the hepatopancreas of two species of aquatic crabs, the blue crab Callinectes sapidus and the stone crab Menippe mercenaria, and two species of land crabs, the blackback land crab Gecarcinus lateralis and the blue land crab Cardisoma guanhumi. Only aquatic crabs showed measurable activities of uricase: 11.5 ± 1.1 nmol urate oxidized per min and per g tissue for C. sapidus (N = 4) and 1.2 ± 0.7 nmol min-1 g-1 (N = 11) for M. mercenaria. These crabs also exhibit a higher XDH activity than XOD activity (approximately 4-5 fold). This is also the case for C. guanhumi but not for G. lateralis which has similar low activities of both enzymes. The possible physiological implications of these results are discussed regarding nitrogenous excretion, blood oxygen transport, and the adaptation of crabs to a terrestrial habitat.

Effect of oxygen pressure on synthesis and export of nitrogenous solutes by nodules of cowpea

Nodules of cowpea plants (Vigna unguiculata (L.) Walp. cv. Vita 3 :Bradyrhizobium CB756) cultured for periods of 23 d with their root systems maintained in atmospheres containing a range of partial pressures of O2 (pO2; 1-80%, v/v, in N2) formed and exported ureides (allantoin and allantoic acid) as the major products of fixation at all pO2 tested. In sub-ambient pO2 (1 and 2.5%) nodules contained specific activities of uricase (urate: O2 oxidoreductase; EC 1.7.3.3) and allantoinase (allantoin hydrolyase; EC 3.5.2.5) as much as sevenfold higher than in those from air. On a cell basis, uninfected cells in nodules from 1% O2 contained around five times the level of uricase. Except for NAD: glutamate synthase (EC 1.4.1.14), which was reduced in sub-ambient O2, the activities of other enzymes of ureide synthesis were relatively unaffected by pO2. Short-term effects of pO2 on assimilation of fixed nitrogen were measured in nodules of air-grown plants exposed to subambient pO2 (1, 2.5 or 5%, v/v in N2) and(15)N2. Despite a fall in total(15)N2 fixation, ureide synthesis and export was maintained at a high level except in 1% O2 where formation was halved. The data indicate that in addition to the structural and diffusional adaptations of cowpea nodules which allow the balance between O2 supply and demand to be maintained over a wide range of pO2, nodules also show evidence of biochemical adaptations which maintain and enhance normal pathways for the assimilation of fixed nitrogen.

Fermentation ofCandida utilis for uricase production

Conditions for the production of microbial uricase byCandida utilis were studied. For the selected strain, hypoxanthine proved to be the most effective inducer of uricase formation. The highest values of biomass as well as uricase activity in the mechanically agitated fermentor were obtained under the following conditions: 50 h, rotation impeller speed 7 s−1, air flow rate 1.25×10−5 m3s−1, concentration of inducer 0.1%.

Purification of Nodule-Specific Uricase From Soybean by Arginine-Sepharose Affinity Chromatography

A method to purify uricase from soybean root nodules is described. The separation uses a single affinity chromatography step on Arginine-Sepharose, which was constructed by coupling L-Arginine to Activated CH-Sepharose 4B. Crude extracts were loaded onto small columns of Arginine-Sepharose and a significant retardation of uricase was observed. With a re-run of the fraction containing maximal uricase activity on the same column highly purified enzyme was obtained. Analysis by SDS-polyacrylamide gel electrophoresis revealed two protein bands. Analytical isoelectric focusing showed two isoforms of uricase, one dominating with pI 9.0 and a minor with pI 7.0.

Elektronenmikroskopisch morphometrische Untersuchung der Entwicklung von Peroxisomen bei gesunden Ferkeln in den ersten 4 Lebenswochen

The number of hepatic peroxisomes, cores, marginal plates, and microperoxisomes in piglets increases from a low level at birth, compared with other species e.g. the rat, to day 28 of life. The number of peroxisomes is correlated to the activity of uricase, but morphometry alone is not sufficient to quantitate uricase activity. These findings might explain the occurrence of renal uric acid deposition (infarction) during a catabolic state, where body protein is metabolized to an increased extent and purine levels rise. Due to a relative lack of uricase uric acid is not completely oxidised to allantoin.

Specialization for ureide biogenesis in the root nodules of black locust (Robinia pseudoacacia L.), an amide exporter

Black locust (Robinia pseudoacacia L.), a member of the legume tribe Robinieae, has indeterminate root nodules and is primarily an amide exporter. However, in greenhouse-grown seedlings inoculated with rhizobia, ureide-N comprises approximately 8% of the total soluble-N in the xylem sap. Ultrastructurally, young interstitial cells (i.e., uninfected cells in the infected region near the nodule meristem) develop enlarged peroxisomes and abundant tubular ER, properties heretofore found to be characteristic only of members of the Phaseoleae, which have determinate nodules and are exporters principally of ureides. Many of the peroxisomes in the interstitial cells of black locust nodules react strongly for uricase (EC 1.7.3.3) activity in a cytochemical test employing diaminobenzidine, but some of the peroxisomes, particularly those farther back from the meristem, react weakly or not at all, even though enlarged. The ultrastructural specializations of the interstitial cells seem disproportionately large in comparison to the relatively low levels of ureides in the xylem transport stream. We suggest that similar specialization of some of the interstitial cells might be found in other legumes that export low amounts of ureides.

Specialization of the inner cortex for ureide production in soybean root nodules

The possibility that cells in the inner cortex of determinate root nodules participate in ureide production from recently fixed N2, as do the uninfected (interstitial) cells of the infected central region, has been investigated in soybean (Glycine max) inoculated as seeds withBradyrhizobium japonicum. Like the interstitial cells, cells of the three innermost cortical layers produce enlarged peroxisomes and a meshwork of tubular ER during differentiation. These changes are most pronounced in the innermost cortical layer, are successively less so in the 2nd and 3rd layers, and are usually undetectable in more distant layers. Peroxisomes in the inner three layers are stained in the DAB (3,3′-diaminobenzidine) test for uricase (EC 1.7.3.3) activity, indicative of the potential for ureide formation, but peroxisomes in more distant cortical cells are not stained. A nodulespecific uricase also is demonstrable in the inner three cortical layers by immunogold labeling enhanced with silver for visualization in the light microscope. The observations suggest that with respect to ureide production the cells of the inner layers of the cortex are functionally similar to the interstitial cells of the infected region despite the apparent distinctiveness of the two regions anatomically.

Use of Polyethylene Glycol-Modified Uricase (PEG-Uricase) to Treat Hyperuricemia in a Patient With Non-Hodgkin Lymphoma

Modification by covalent attachment of monomethoxypolyethylene glycol (PEG) can reduce the immunogenicity and prolong the circulating life of injected enzymes, making their use as therapeutic agents feasible. We report the first clinical use of PEG-modified Arthrobacter protoformiae uricase (PEG-uricase) to treat hyperuricemia in a patient with non-Hodgkin lymphoma and renal insufficiency who was allergic to allopurinol. Two intramuscular injections totaling 3 U/kg body weight during the first 30 hours of treatment lowered the plasma urate level from 910 to 190 mumol/L (15.3 to 3.2 mg/dL), after which a dose of 2 U/kg every 5 to 6 days maintained the plasma urate level at 540 mumol/L (9 mg/dL) or lower. After the injection of PEG-uricase, uricase activity appeared in plasma rapidly, peaking within 24 hours and persisting for approximately 5 days; an inverse relation between plasma uricase activity and plasma urate concentration was noted. The agent was nontoxic and well tolerated. No antibody to either PEG-uricase or unmodified uricase developed over a 3-week period, during which four doses of PEG-uricase were administered. Because of its long circulating life, PEG-uricase is probably a more effective hypouricemic agent than unmodified uricase, which has previously had limited use. As an adjunct to cytolytic therapy for hematologic malignancies when protection from hyperuricemia is needed rapidly, PEG-uricase deserves further study.

A human genomic sequence highly homologous to the 3′-untranslated region of rat uricase mRNA

A human genomic sequence was isolated from a library using a rat uricase cDNA probe. Sequence analysis has shown that it is highly homologous to the 3'-untranslated region of rat uricase mRNA. Total loss of uricase activity in human is, therefore, not due to total loss of the gene. Discovery of high degree of conservation of the non-coding region of the gene would be of great interest as we attempt to learn the process of gene evolution.

Isolation and enzymological characterization of infected and uninfected cell protoplasts from root nodules of Glycine max

Infected and uninfected cell protoplasts were isolated from soybean (Glycine max L. Merr. cv. Akisengoku) root nodules and purified by the use of nylon mesh filters and discontinuous Percoll gradients. Activities of the enzymes involved in carbon and nitrogen metabolism were measured in cytoplasmic fractions of purified protoplasts, as well as in the bacteroids isolated from infected cell protoplasts and in the cortical tissues after enzymatic digestion of the central zone of the nodules.A high degree of purity of both infected and uninfected cells was demonstrated by microscopic observations and assays of β‐hydroxybutyrate dehydrogenase (EC 1.1.1.30) and uricase (EC 1.7.3.3) activities and leghemoglobin contents.As a whole, higher specific activities of enzymes of glycolysis were found in the cortical and uninfected cells than in the infected cells. The activities of glycolytic enzymes were extremely low in the bacteroids. Invertase (EC 3.2.1.26) was highly localized in the cortex. However, activity of sucrose synthase (EC 2.4.1.13) was highest in the cytosol of infected cells. Alcohol dehydrogenase (EC 1.1.1.1) and lactate dehydrogenase (EC 1.1.1.27) activities were much higher in uninfected than in infected cells. Specific activities of enzymes for nitrogen assimilation, that is, glutamine synthetase (EC 6.3.1.2), glutamate synthase (EC 1.4.1.14), aspartate (EC 2.6.1.1) and alanine (EC 2.6.1.2) aminotransferase were several‐fold higher in uninfected cells than in the infected cells.The results are discussed in relation to the possible cellular organization of carbon and nitrogen metabolism in soybean root nodules.

Localization of uricase in mycorrhizas of Ophrys lutea Cav.

Infected roots of Ophrys lutea present nymerous peroxisome profiles identified by diaminobenzidine (DAB) staining. The presence of uricase in these peroxisomes could also be recognized cytochemically. The activities of catalase and uricase, assayed using a crude fraction of peroxisomes, reached 0·8 μkat min-1 protein-1 and 1·73 nakat min-1 mg protein-1 respectively. The role of uricase in purine metabolism is discussed in terms of host/endophyte interactions in O. lutea mycorrhzas.