Structural and expression analysis of uricase mRNA from Lotus japonicus

Uricase (nodulin-35) cDNA, LjUr, was isolated from nodules of a model legume, Lotus japonicus. LjUr expression was most abundant in nodules, although it was detected in nonsymbiotic tissues as well, particularly in roots. Expression in nodules was detected in uninfected cells, nodule parenchyma, and, more intensely, in vascular bundles. Phylogenetic analysis of uricase sequences from various legumes indicated that uricases of amide- and ureide-transporting legumes form two distinct clades. LjUr is in the cluster of amide-transport legumes even though L. japonicus bears determinate nodules.     

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.     

Immunogold localization of nodule-specific uricase in developing soybean root nodules

Immunogold labeling was used to study the time of appearance and distribution of a nodule-specific form of uricase (EC 1.7.3.3) in developing nodules of soybean (Glycine max (L.) Merr.) inoculated with Bradyrhizobium japonicum. The enzyme was detected in thin sections of tissue embedded in either L R White acrylic resin or Spurr's epoxy resin, by employing a polyclonal antibody preparation active against a subunit of soybean nodule uricase. Antigenicity was better preserved in L R White resin, but ultrastructure was better maintained in Spurr's. Uricase was first detectable with protein A-gold in young, developing peroxisomes in uninfected cells, coincident with the release of Bradyrhizobium bacteroids from infection threads in adjacent infected cells. As the peroxisomes enlarged, labeling of the dense peroxisomal matrix increased. Gold particles were never observed over the paracrystalline inclusions of peroxisomes, however. Despite a close association between enlarging peroxisomes and tubular endoplasmic reticulum, uricase was not detectable in the latter. In mature nodules, labeling of uricase was limited to the large peroxisomes in uninfected cells. Small peroxisome-like bodies present in infected cells did not become labeled.Abbreviations BSA bovine serum albumin - Da dalton - ER endoplasmic reticulum - IgG immunoglobulin G     

Oxygen regulation of uricase and sucrose synthase synthesis in soybean callus tissue is exerted at the mRNA level

The effect of lowering oxygen concentration on the expression of nodulin genes in soybean callus tissue devoid of the microsymbiont has been examined. Poly(A)⁺ RNA was isolated from tissue cultivated in 4% oxygen and in normal atmosphere.Quantitative mRNA hybridization experiments using nodule-specific uricase (Nodulin-35) and sucrose synthase (Nodulin-100) cDNA probes confirmed that the synthesis of the uricase and sucrose synthase is controlled by oxygen at the mRNA level.The steady-state levels of uricase and sucrose synthase mRNA increased significantly (5–6- and 4-fold respectively) when the callus tissue was incubated at reduced oxygen concentration. Concomitant with the increase in mRNA level a 6-fold increase in specific activity of sucrose synthase was observed.Two messengers representing poly-ubiquitin precursors also responded to lowering the oxygen concentration. The increase was about 5-fold at 4% oxygen. No expression at atmospheric oxygen or in response to low oxygen was observed when using cDNA probes for other nodulin genes such as leghemoglobin c₃, nodulin-22 and nodulin-44.     

Identification of uricase as a potential target of plant thioredoxin: Implication in the regulation of nodule development

During symbiotic nodule development in legume roots, early signaling events between host and rhizobia serve critical determinants for the proper onset of nodule morphogenesis, nitrogen fixation, and assimilation. Previously we isolated thioredoxin from soybean nodules as one of differentially expressed genes during nodulation and noted its positive role in nitrogen fixation. To identify the target proteins of thioredoxin in nodules, we used thioredoxin affinity chromatography followed by mass spectrometry. Nodulin-35, a subunit of uricase, was found to be a target of thioredoxin. Their interaction was confirmed by pull-down assay and by bimolecular fluorescent complementation. With an increased uricase activity observed also in the presence of thioredoxin, these results appear to implicate a novel role of thioredoxin in the regulation of enzyme activities involved in nodule development and nitrogen fixation.     

Expression of antisense nodulin-35 RNA in Vigna aconitifolia transgenic root nodules retards peroxisome development and affects nitrogen availability to the plant

A nodulin-35 (N-35) cDNA encoding nodule-specific uricase (EC 1.7.3.3.) was isolated from a Vigna aconitifolia (mothbean) root nodule cDNA library. Sequence analysis of Vigna uricase (VN-35) cDNA revealed 90% homology to that of soybean. The VN-35 cDNA was inserted in the antisense orientation downstream of the CaMV—35S promoter, and transgenic hairy roots were formed on Vigna plants using Agrobacterium rhizogenes . Infection with Bradyrhizobium (cowpea) gave rise to root nodules on transgenic hairy roots supported by the wild-type shoot. Expression of antisense VN-35 RNA was detected in transgenic nodules on individual roots using polymerase chain reaction (PCR). The nodules expressing antisense VN-35 RNA were smaller in size and showed lower uricase activity than nodules formed on the hairy roots transformed with a binary vector containing β-glucuronidase (GUS) gene (used as control), and the plants exhibited nitrogen deficiency symptoms. Ultrastructural analysis and immunogold labeling with antibody against soybean N-35 revealed that the growth of peroxisomes was retarded in transgenic nodules expressing antisense VN-35 RNA. These data suggest that a reduction in ureide biosynthesis limits the availability of symbiotically reduced nitrogen to the plant. The nodules of tropical legumes appear to be specialized in nitrogen assimilation and are developmentally controlled to produce and transport ureides.     

Regulation of uricase activity in developing roots of Glycine max, non-nodulating variety A62-2

Appearance of uricase activity in radicles in the early stageof seed germination of soybean, non-nodulating variety A62–2,was coincident with the increase in contents of uricase cofactor.With aging, the radicles lost uricase activity without a decreaseof uricase apoprotein, indicating the decrease of uricase cofactor. The uricase cofactor could be produced in aerobic conditions.The uricase cofactor was degraded by a protein having maximumactivity at pH 6.5. The protein participating in the cofactordegradation could not be detected in 1 or 2-day-old seedlings,but was detected in the later stage. Uricase apoprotein was present in all the developmental stagesof soybean root in spite of the absence of uricase activity.When the mature stem of a soybean plant, non-nodulating variety,was decapitated, uricase activity was elicited in the rootsin accordance with the increase in the level of the uricasecofactor. (Received August 19, 1976; )     

Cellular compartmentation of ureide biogenesis in root nodules of cowpea (Vigna unguiculata (L.) Walp.)

Cowpea (Vigna unguiculata (L.) Walp.) nodules have been investigated by means of cytochemical and immunocytochemical procedures at the ultrastructural level in order to assess the role of the uninfected cells in ureide biogenesis. Uricase activity in the nodules was shown by cytochemical methods to be localized exclusively in the numberous large peroxisomes confined to the uninfected cells; the small peroxisomes in the infected cells did not stain for uricase. Uricase was also localized in the peroxisomes of uninfected cells by immunogold techniques employing polyclonal antibodies against nodule-specific uricase of soybean. There was no labeling above background of any structures in the infected cells. The results indicate that the uninfected cells are essential for ureide biogenesis in cowpea. Although tubular endoplasmic reticulum, the presumptive site of allantoinase, increases greatly in the uninfected cells during nodule development, it virtually disappears as the nodules mature. The inconsistency between the disappearance of the tubular endoplasmic reticulum from older nodules and the high allantoinase activity reported for older plants remains to be explained.Abbreviations DAB 3,3-diaminobenzidine - ER endoplasmic reticulum - GARG goat anti-rabbit immunoglobulin G - IgG immunoglobulin G - kDa knodalton - Mr apparent molecular mass - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Expression of nodule-specific uricase in soybean callus tissue is regulated by oxygen

In soybean root nodules the enzyme uricase is expressed concomitantly with nodule development. The initial expression of this protein does not depend on active nitrogen fixation, as demonstrated by analysis of uricase activity in effective and ineffective root nodules. However, the maximal level of uricase activity is determined by the infecting Rhizobium japonicum strain. Sterile root cultures and callus tissue, devoid of the microsymbiont, were incubated at varying oxygen concentrations and analyzed for uricase activity. The specific activity of uricase was increased by lowering the oxygen concentration, with the highest activity obtained around 4−5% oxygen. The increase in uricase activity was due to increased uricase synthesis, as demonstrated by in vivo labelling of callus culture followed by immunoprecipitation with antibodies raised against highly purified nodule uricase.     

A Cytosolic Thioredoxin Acts as a Molecular Chaperone for Peroxisome Matrix Proteins as Well as Antioxidant in Peroxisome

Thioredoxin (TRX) is a disulfide reductase present ubiquitously in all taxa and plays an important role as a regulator of cellular redox state. Recently, a redox-independent, chaperone function has also been reported for some thioredoxins. We previously identified nodulin-35, the subunit of soybean uricase, as an interacting target of a cytosolic soybean thioredoxin, GmTRX. Here we report the further characterization of the interaction, which turns out to be independent of the disulfide reductase function and results in the co-localization of GmTRX and nodulin-35 in peroxisomes, suggesting a possible function of GmTRX in peroxisomes. In addition, the chaperone function of GmTRX was demonstrated in in vitro molecular chaperone activity assays including the thermal denaturation assay and malate dehydrogenase aggregation assay. Our results demonstrate that the target of GmTRX is not only confined to the nodulin-35, but many other peroxisomal proteins, including catalase (AtCAT), transthyretin-like protein 1 (AtTTL1), and acyl-coenzyme A oxidase 4 (AtACX4), also interact with the GmTRX. Together with an increased uricase activity of nodulin-35 and reduced ROS accumulation observed in the presence of GmTRX in our results, especially under heat shock and oxidative stress conditions, it appears that GmTRX represents a novel thioredoxin that is co-localized to the peroxisomes, possibly providing functional integrity to peroxisomal proteins.     

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.     

Purification and characterization of uricase,a nitrogen-regulated enzyme,from Neurospora crassa

Uricase, a purine catabolic enzyme, is controlled by induction and by nitrogen catabolite repression in Neurospora. Uricase was purified nearly 1000-fold to homogeneity. Only a single protein band could be detected in analytical gels of the pure enzyme, and the protein band in each case corresponded exactly to the position of in situ staining for enzyme activity. The molecular weight of native uricase was estimated to be 123,000 ± 7000. The enzyme is a tetramer composed of identical or similar-sized subunits. The K_m value of uricase for uric acid was estimated to be 4.2 × 10^?5, m. Oxonic acid was shown to be a competitive inhibitor of uricase, with a K_i value of 6.7 × 10^?7, m. Uricase is a stable enzyme and is not subject to feedback inhibition by ammonia, glutamate, or glutamine in Neurospora. The regulation of uricase appears to occur primarily at the biosynthesis level. Uricase appears to be a metalloenzyme with no essential sulfhydryl groups.     

Isolation and characterization of uricase from bean leaves and its comparison with uredospore enzymes

Uricase (urate: oxygen oxidoreductase; EC 1.7.3.3) from bean (Phaseolus vulgaris) leaves and uredospores of two different rust fungi (Uromyces phaseoli and Uromyces fabae) has been purified to electrophoretic homogeneity by a procedure which includes xanthine–agarose affinity chromatography as the main step. Pure preparations had similar specific activities (2–6 U mg⁻¹) with turnover numbers from 250 to 750 min⁻¹, and all enzymes were tetramers consisting of identical or similar-sized subunits of 32–33 kDa. They also exhibited similar optimum pH (around 9.0), showed hyperbolic kinetics with K_m values of 15–34 μM and behaved similarly against a number of putative activators/inhibitors, all of them being inhibited only by oxonate and xanthine. However, leaf and uredospore uricases differed in the strength of binding to DEAE-cellulose since leaf uricase did not bind to the exchanger and that from U. fabae bound stronger than that of U. phaseoli. Uredospore uricases showed complete antigenic independence against anti-uricase polyclonal antibodies from bean leaves and anti-uricase monoclonal antibodies from soybean nodules. Cross-reaction was observed between leaf uricase and nodule monoclonal antibodies and between nodule enzyme and leaf polyclonal antibodies. These results confirm the homogeneity of plant uricases and demonstrate that fungal obligate parasites have their own uricase, which is similar to the plant enzyme in many molecular and kinetic properties but different in DEAE-cellulose binding characteristics and immunological properties.     

Modification of yeast uricase with polyethylene glycol: disappearance of binding ability towards anti-uricase serum

Uricase from Candida utilis was modified with activated polyethylene glycol (2-O-methoxypolyethylene glycol-4,6-dichloro-s-triazine) of molecular weight of 5,000 daltons. The modification of 43% of the total amino groups in the uricase molecule gave rise to a complete loss of the binding ability towards anti-uricase serum from rabbit. This modified uricase retained 15% of the enzymic activity of non-modified uricase.     

Regulation of expression of the Rhizopus oryzae uricase and urease enzymes

The regulation of intracellular urease and uricase activities was examined in Rhizopus oryzae. Urease activity (2.4 U/mg protein) was present in R. oryzae mycelium grown in minimal medium containing NH4CI as sole nitrogen source. This activity increased threefold under nitrogen derepression conditions, but no induction by urea was detected. Control of urease activity in R. oryzae differs from that found in Neurospora crassa but resembles the situation in Aspergillus nidulans. No uricase activity was detected in R. oryzae mycelium grown in minimal medium containing NH4Cl as sole nitrogen source. Uricase activity was increased 10- to 40-fold under derepression conditions and was induced by exogenous uric acid (60- to 78-fold). Control of the R. oryzae uricase differs from that found in N. crassa and A. nidulans. This is the first analysis of the regulation of enzymes from the purine catabolic pathway in any member of the Zygomycetes.     

Postnatal development of hepatic uricase and peroxisomes in baby pigs

1. The activity of uricase, the densities of peroxisomes and cores in liver samples of baby pigs up to 4 weeks of age were investigated. 2. From 1 to 4 weeks of age, uricase activity as well as counts of cores and peroxisomes increased 5.5-, 3.3- and 2-fold. 3. Uricase activity and counts of cores and peroxisomes were correlated (P less than 0.001) with age in linear relationships. 4. Calculated for time of birth uricase activity was very low and ratio of cores to peroxisomes was 1:7. 5. From 0 to 28 days of age the calculated increases of uricase activity and counts of cores and peroxisomes were 178-, 10- and 3-fold.