Enzymes of purine catabolism in soybean plants

Remarkable formation and utilization of allantoin is observedin soybean (Glycine max variety A62-1). To study this, variousenzymes involved in purine catabolism (i.e., xanthine oxidase,uricase and allantoinase) were measured in different regionsof soybean plants during development. Uricase, which catalyzesthe direct formation of allantoin from uric acid, was studiedin detail. The activities of these three enzymes were highest in the rootnodules, indicating that the nodules are the major site of allantoinmetabolism. Radicles only showed appreciable activity about80 hr after the seeds were planted. Allantoinase activity wasdetected in all regions tested, showing that allantoin translocatedfrom the nodules can be metabolized in the roots, stem and leaves.In the nodules, xanthine oxidase was localized in the nuclearfraction, while uricase was mainly restricted to the mitochondrialfraction and allantoinase to the soluble fraction. Uricase was partially purified from the nodules and radicles,respectively. The pH optimum of enzyme from the nodules was9.5, whereas that of enzyme from the radicles was 7.0. The enzymefrom the nodules did not require a cofactor, while that fromthe radicles showed an absolute requirement for a cofactor,which was a low molecular substance easily separable from theapoprotein. Thus, the uricase in nodules differs in chemicalproperties from that in the host plant. The results are discussedin relation to change in the allantoin level in soybean tissues. (Received November 1, 1974; )     

Characteristics of a Urate-Degrading Diamine Oxidase-Peroxidase Enzyme System in Soybean Radicles

The cadaverine content of soybean radicles showed a maximumpeak 3–4 days after planting. The variation coincidedwith radicle uricase activity during seed germination. The uricase activity could not be fractionate when the bufferpH for the extraction was at 6.0. The addition of 1 M KCl orNaCl to the buffer allowed the extraction of the uricase activity,but an addition of 1 M MgCl₂ or BaCl₂ inhibited this enzyme'sactivity. The urate-degrading enzyme system was purified 248-fold permilligram of protein from soybean radicles. The respective K_mvalues of the diamine oxidase activity for cadaverine and ofthe urate-degrading activity for hydrogen peroxide and uratewere 1.25, 2.93 and 50.3 µM. Analysis by gel electrophoresisof the partially purified enzyme fraction revealed that theurate-degrading enzyme system consisted of a peroxidase thatdegrades urate with hydrogen peroxide and a diamine oxidasethat releases hydrogen peroxide. These data are evidence that a urate-degrading diamine oxidaseand peroxidase system exists in soybean radicles and that thereaction rate of urate-degradation is controlled by the concentrationof cadaverine. (Received November 28, 1984; Accepted April 8, 1985)     

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.     

Relevance of weak flavin binding in human D‐amino acid oxidase

In the brain, the human flavoprotein D ‐amino acid oxidase (hDAAO) is involved in the degradation of the gliotransmitter D ‐serine, an important modulator of NMDA‐receptor‐mediated neurotransmission; an increase in hDAAO activity (that yields a decrease in D ‐serine concentration) was recently proposed to be among the molecular mechanisms leading to the onset of schizophrenia susceptibility. This human flavoenzyme is a stable homodimer (even in the apoprotein form) that distinguishes from known D ‐amino acid oxidases because it shows the weakest interaction with the flavin cofactor in the free form. Instead, cofactor binding is significantly tighter in the presence of an active site ligand. In order to understand how hDAAO activity is modulated, we investigated the FAD binding process to the apoprotein moiety and compared the folding and stability properties of the holoenzyme and the apoprotein forms. The apoprotein of hDAAO can be distinguished from the holoenzyme form by the more “open” tertiary structure, higher protein fluorescence, larger exposure of hydrophobic surfaces, and higher sensitivity to proteolysis. Interestingly, the FAD binding only slightly increases the stability of hDAAO to denaturation by urea or temperature. Taken together, these results indicate that the weak cofactor binding is not related to protein (de)stabilization or oligomerization (as instead observed for the homologous enzyme from yeast) but rather should represent a means of modulating the activity of hDAAO. We propose that the absence in vivo of an active site ligand/substrate weakens the cofactor binding, yielding the inactive apoprotein form and thus avoiding excessive D ‐serine degradation.     

Urate-mediated regulation of urate oxidase inChlamydomonas reinhardtii

Summary Expression of uncase (urate oxidase) fromChlamydomonas reinhardtii has been investigated by using specific polyclonal antibodies. By Western blot analyses performed under nondenaturing conditions, a 124 kDa protein band corresponding to active uricase was detected in protein extracts from cells cultured with urate or nitrogen-starved cells. This protein band was absent in cells cultured with ammonium. Besides the 124 kDa band, the antibodies also reacted with a massive protein band, with an apparent molecular mass of 500 kDa, that was detected in all nutritional conditions assayed. In vitro, inactive uricase from cells grown with ammonium was activated by incubation in presence of urate. The appearance of uricase activity in vitro coincided with a decrease of the 500 kDa protein and an increase of the 124 kDa band corresponding to the active enzyme. We suggest that a posttranslational regulation of uricase synthesis takes place inC. reinhardtii, and that urate may be responsible for the assembly or maturation of inactive precursors to form the active uricase.     

Nodulin-35: a subunit of specific uricase (uricase II) induced and localized in the uninfected cells of soybean nodules

Nodulin-35, a protein specific to soybean root nodules, was purified under non-denaturing conditions (DEAE-cellulose followed by Sephacryl S-200 chromatography) to homogeneity. The holoprotein showed uricase (EC 1.7.3.3) activity. Analytical ultracentrifugation under non-denaturing conditions revealed a molecule of 124 kd, S°_20W = 8.1; however, under denaturing conditions a value of 33 kd, S°_20W = 1.9, was obtained. This indicated that nodulin-35 is the 33-kd subunit of a specific soybean root nodule uricase (uricase II) and that the enzyme contains four similar subunits. The native molecule contains ˜1.0 mol Cu²⁺ per mol, has an isoelectric point of ˜9.0 and a pH optimum for uricase activity at 9.5. Uricase activity found in young uninfected soybean roots is due to another form of enzyme (uricase I) which is of ˜190 kd, has maximum activity at pH 8.0 and does not contain any subunit corresponding in size to nodulin-35. Uricase I, also present in young infected roots, declines at a time when nodulin-35 appears. Monospecific antibodies prepared against uricase II (nodulin-35) showed no cross-reactivity. Uricase II was localized in the uninfected cells of the nodule tissue. These results are consistent with the concept that a nodule-specific ureide metabolism takes place in peroxisomes of uninfected cells, and suggest the participation of uricase II in this pathway.     

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.     

Cadaverine Involved in Urate Degrading Activity (Uricase Activity) in Soybean Radicles

Cadaverine, a 5-carbon diamine, was identified as the cofactorof uricase activity previously found in soybean seedlings. Thesubstance purified from freeze dried hypocotyls was subjectedto liquid chromatography, mass spectrometry, ¹H- and ¹³C-nuclearmagnetic resonance spectrometry for identification. The concentrationsof cadaverine in 3-day-old radicles and hypocotyls were 2.37mM and 5.09 mM, respectively. Other polyamine concentrationswere low. Biogenic polyamines (cadaverine, putrescine, spermidineand spermine) functioned as cofactors, whereas conjugated polyamines(tyramine and histamine) and amino acids had no effect. Theaddition of catalase to the assay system counteracted the effectof cadaverine. Peroxide at appropriate concentrations actedlike cadaverine with an identical K_m value, suggesting thaturate degrading activity can be ascribed to the diamine oxidase-peroxidasesystem. (Received October 19, 1982; Accepted December 23, 1982)     

In vitro reconstitution of NADH: nitrate reductase in nitrate reductase-deficient mutants of barley

Summary In vitro complementation of the nitrate reductase-deficient barley mutant nar2a extracts with molybdenum cofactor from commercial xanthine oxidase resulted in reactivation of NADH: nitrate reductase activity. Maximum reactivation was achieved with 7.5 g/ml xanthine oxidase (final concentration), 10 mM glutathione (final concentration) and incubation for 30 min at room temperature (ca. 25°C). This in vitro complementation assay was used to determine the presence of functional apoprotein and molybdenum cofactor in 12 nitrate reductase-deficient barley mutants. Extracts of all nar1 alleles contained functional molybdenum cofactor (complemented with nar2a) but they lacked functional apoprotein (did not complement with molybdenum cofactor from xanthine oxidase). The nar2a, nar3a and nar3b extracts were able to donate functional apoprotein, but were poor sources of functional molybdenum cofactor. These data are in agreement with our previous assignment of nar1 to the barley NADH: nitrate reductase structural locus and nar2 and nar3 to molybdenum cofactor functions. Wild type cv. Steptoe barley seedlings grown in the absence of nitrate and lacking nitrate reductase activity contained low levels of molybdenum cofactor. Nitrate induction resulted in a several-fold increase in the measurable molybdenum cofactor levels that was correlated with the increase in nitrate reductase activity.Scientific Paper No. 6839. College of Agriculture Research Center, Washington State University, Pullman. Project Nos. 0430 and 0233. This work was supported in part by National Science Foundation Grant PCM 81-19096 and USDA Competitive Research Grant 82-CRCR-1-1112     

Appearance of purine-catabolizing enzymes in fix and fix root nodules on soybean and effect of oxygen on the expression of the enzymes in callus tissue

The appearance of enzymes involved in the formation of ureides, allantoin, and allantoic acid, from inosine 5′-monophosphate was analyzed in developing root nodules of soybean (Glycine max). Concomitant with development of effective nodules, a substantial increase in specific activities of the enzymes 5′-nucleotidase (35-fold), purine nucleosidase (10-fold), xanthine dehydrogenase (25-fold), and uricase (200-fold), over root levels was observed. The specific activity of allantoinase remained constant during nodule development. With ineffective nodules the activities were generally lower than in effective nodules; however, the activities of 5′-nucleotidase and allantoinase were 2-fold higher in ineffective nodules unable to synthesize leghemoglobin than in effective nodules. Since the expression of uricase has been shown to be regulated by oxygen (K Larsen, BU Jochimsen 1986 EMBO J 5: 15-19), the expression of the remaining enzymes in the purine catabolic pathway were tested in response to variations in O₂ concentration in sterile soybean callus tissue. Purine nucleosidase responded to this treatment, exhibiting a 4-fold increase in activity around 2% O₂. 5′-Nucleotidase, xanthine dehydrogenase, and allantoinase remained unaffected by variations in the O₂ concentration. Hence, the expression of two enzymes involved in ureide formation, purine nucleosidase and uricase, has been demonstrated to be influenced by O₂ concentration.     

Enhancement of a Novel Extracellular Uricase Production by Media Optimization and Partial Purification by Aqueous Three-Phase System

Uricase (urate: oxygen oxidoreductase, EC 1.7.3.3), an enzyme belonging to the class of oxidoreductases, catalyzes the enzymatic oxidation of uric acid to allantoin and finds a wide variety of application as therapeutic and clinical reagent. In this study, uricase production ability of the bacterial strains isolated from deep litter poultry soil is investigated. The strain with maximum extracellular uricase production capability was identified as Xanthomonas fuscans subsp. aurantifolii based on 16S rRNA sequencing. Effect of various carbon and nitrogen sources on uricase productivity was investigated. The uricase production for this strain was optimized using statistically based experimental designs and resulted in uricase activity of 306 U/L, which is 2 times higher than initial uricase activity. Two-step purification, such as ammonium sulfate precipitation and aqueous two-phase system, was carried out and a twofold increase in yield and specific activity was observed.     

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.     

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     

Biochemical characterization of nitrate and nitrite reduction in the wild-type and a nitrate reductase mutant of soybean

Enzyme activities involved in nitrate assimilation were analyzed from crude leaf extracts of wild-type (cv. Williams) and mutant ( nr₁ ) soybean [ Glycine max (L.) Merr.] plants lacking constitutive nitrate reductase (NR) activity. The nr₁ soybean mutant (formerly LNR-2), had decreased NADH-NR, FMNH₂-NR and cytochrome c reductase activities, all of which were associated with the loss of constitutive NR activity. Measurement of FMNH₂-NR activity, by nitrite determination, was accurate since nitrite reductase could not use FMNH₂ as a reductant source. Nitrite reductase activity was normal in the nr₁ plant type in the presence of reduced methyl viologen. Assuming that constitutive NR is similar in structure to nitrate reductases from other plants, presence of xanthine dehydrogenase activity and loss of cytochrome c reductase activity indicated that the apoprotein and not the molybdenum cofactor had been affected in the constitutive enzyme of the mutant. Constitutive NR from urea-grown wild-type plants had 1) greater ability to use FMNH₂ as an electron donor, 2) a lower pH optimum, and 3) decreased ability to distinguish between NO₃⁻ and HCO₃⁻, compared with inducible NR from NO₃⁻-grown nr₁ plants. The presence in soybean leaves of a nitrate reductase with a pH optimum of 7.5 is contrary to previous reports and indicates that soybean is not an exception among higher plants for this activity.     

Effect of surfactants and inducers on increased uricase production under submerged fermentations by Bacillus cereus

Uricase is a clinical enzyme used for the oxidation of uric acid crystals in gout disease. The present study aimed to increase the suitable surfactant-mediated uricase production on induction by different concentrations of inducers. The efficiency of Bacillus cereus to produce extracellular uricase enzyme was studied in uric acid-containing agar plates. Among the studied inducers, uric acid is the potential inducer for uricase production under submerged fermentations (SMF), which induced 19.41?U/ml uricase in medium containing 2.0?g/L of uric acid, however further increase in the uric acid concentration decreased uricase production, which could be because of substrate inhibition. The physical parameters including agitation speed (rpm) and time duration (h) of uricase production were optimized and found to produce optimum uricase at 150?rpm in 26?h of SMF. Among the studied surfactants, nonionic surfactant, polyvinyl alcohol has shown a remarkable increase in the uricase production of 31.58?U/ml, which is a 61% increase under optimized conditions in SMF. The stability of produced uricase was found at pH 7.5 and temperature 30°C. Also the effects of various metal ions (1?mM) on the uricase activity were studied and observed to be inhibitory in nature in the descending order K⁺?>?Ca²⁺?>?Zn²⁺?>?Fe³⁺?>?Ni²⁺?>?Mg²⁺?>?Mn²⁺?>?Cu²⁺.     

Complementation analysis of a nitrate reductase deficient Hyoscyamus muticus cell line by somatic hybridisation

Summary Complementation of a nitrate reductase deficient variant of Hyoscyamus muticus (MA-2) and nitrate reductase apoenzyme (nia-115) and cofactor mutants (cnx-68) of Nicotiana tabacum was studied by protoplast fusion. Selection of prototrophic intergeneric somatic hybrids was achieved in combination of MA-2 with the apoenzyme mutant nia-115 of N. tabacum. The H. muticus MA-2 line was therefore classified to be a cnx type variant possessing an altered molybdenum cofactor of the nitrate reductase enzyme complex but unaffected in the apoprotein of nitrate reductase. The nitrate reductase deficient and chlorate resistant characters of MA-2 were functionally coupled recessive traits. Nitrate reductase activity accompanied by chlorate sensitivity could be detected only under inductive conditions in the somatic hybrids. The inductive expression of nitrate reductase in the somatic hybrids arising from the combination of cells harbouring either the inductive or constitutive type nitrate reductase is discussed.Abbreviations DTT 1,4-Dithio-DL-threitol - Mo-co molybdenum containing cofactor - PEG polyethylene glycol     

大豆类受体蛋白激酶基因GmRLCK的克隆及初步分析

植物类受体蛋白激酶(receptor-likeproteinkinase,RLK)在高等植物生长发育和环境刺激的信号传导中起着重要的作用。本文报告了一个新的大豆类受体蛋白激酶基因的全长cDNA克隆及对其基因结构和功能的初步分析。研究表明该基因序列编码的蛋白包含一个跨膜域、一个具有丝氨酸/苏氨酸激酶活性的胞内域和一个缺少N-末端信号肽的胞外域。采用生物信息学方法分析表明,该基因与一些拟南芥菜类受体蛋白激酶基因具有很高的相似性,这些激酶N-末端都缺少信号序列,属于植物胞质类受体激酶(receptor-likecytoplasmickinase,RLCK)亚家族。因此命名该大豆基因为GmRLCK(GenBankAccessionNo.AY687390)。对GmRLCK激酶域中磷酸化可能性较高的位点进行了预测。RT-PCR的结果表明,GmRLCK在大豆子叶、根、花以及豆荚中都有较高的表达,而在胚根、茎和成熟叶片中的表达相对较弱。进化分析表明GmRLCK与一些衰老相关的植物类受体蛋白激酶具有较近的亲缘关系。