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1.
1. The effects of 19 bivalent cations on the activity, stability and capacity to hydrolyse endogenous arginine of axolotl liver arginase were studied. 2. It was found that Fe2+, Co2+, Ni2+ and Zn2+, as well as Mn2+, stabilize the enzyme and render it able to hydrolyse endogenous arginine. 3. By using different concentrations of Co2+ and Ni2+ it was possible to dissociate the effect of each of the metal ions on the activity and stability of arginase from that on its capacity to hydrolyse endogenous arginine. 4. The effects of Mn2+, Co2+ and Ni2+ on the activity and stability of arginase in the homogenate were also observed with purer preparations of the enzyme. 5. It is suggested that the capacity to hydrolyse endogenous arginine is a consequence of the integration of arginase with the arginine-formation sites.  相似文献   

2.
1. Carbamoyl phosphate synthetase, ornithine transcarbamoylase, the arginine-synthetase system and arginase were measured in the livers of ammoniotelic, ureotelic and uricotelic animals. The chelonian reptiles, whose nitrogen excretory patterns vary according to the habitat, and the Mexican axolotl, a neotenic species, were also studied. 2. The levels of the activities of the first three enzymes mentioned correlate with the amount of nitrogen excreted as urea. 3. The terrestrial turtle, which excretes mainly uric acid, maintains a high arginase activity but has very low levels of the activities of the other three enzymes. 4. The first three enzymes of the urea cycle vary in the phylogenic scale in a co-ordinated manner, which suggests that they are under the same regulatory mechanism. 5. Urea formation from endogenous arginine in vitro has a low efficiency in the Mexican axolotl. 6. The induction of metamorphosis in the Mexican axolotl by the administration of l-tri-iodothyronine, which causes a shift from ammonio-ureotelism to complete ureotelism, is accompanied by an increase mainly in carbamoyl phosphate synthetase and also by an improvement in the efficiency of hydrolysis of endogenous arginine in vitro to give urea. 7. The results obtained by differential centrifugation of the urea-cycle enzymes in rat and Mexican-axolotl livers are presented. The location requirements for the integration of a metabolic cycle are discussed.  相似文献   

3.
The syntheses of arginase and ornithine transaminase were studied in two strains ofSaccharomyces cerevisiae, viz. strain B and strain α-Σ1278b. Derepression of both enzymes during nitrogen starvation was shown only by strain B, non-specific induction of arginase only by strain α-Σ1278b. This different response of both strains studied reveals substantial differences in the regulation of enzyme synthesis among yeast strains of one and the same species. The specific enzyme activities observed in chemostat cultures with arginine as the nitrogen source and different sugars, at variable carbon to nitrogen ratios, did not indicate the involvement of carbon catabolite repression in the regulation of arginase and ornithine transaminase syntheses. Specific arginase activities observed in the continuous cultures varied widely and did not show a correlation with the intracellular arginine concentration. Extracellular steady-state arginine concentrations higher than about 0.1mm, in addition to abundant energy supply, were found to be required for high production of arginase. It is suggested that, besides intracellular arginine, extracellular arginine may provide an induction signal necessary for full-scale induction of arginase synthesis. A possible intermediary role of arginine permeases or of other membrane proteins is discussed.  相似文献   

4.
It has been found that, in Neurospora crassa, arginine synthesized from exogenous citrulline was not as effectively hydrolyzed as exogenous arginine. This was explained by the observed inhibition of arginase in vitro and in vivo by citrulline. The high arginine pool formed from exogenous citrulline feedback inhibits the arginine pathway. These two factors allow exogenous citrulline to be used adventitiously and efficiently as an arginine source. Finally, it was found that ornithine was a strong inhibitor of arginase. This suggests that the characteristically high ornithine pool of minimal cultures of Neurospora may act to control a potentially wasteful catabolism of endogenous arginine by arginase.  相似文献   

5.
Arginase is an enzyme that catalyses the hydrolysis of arginine to urea and ornithine. It is abundantly present in the liver of ureotelic animals (i.e. those whose excretion is characterized by the excretion of uric acid as the chief end-product of nitrogen metabolism), but its purification has hitherto not been simple, and the yield not high. Starting with a partially truncated cDNA for human liver arginase recently made available, we constructed an expression plasmid that had tandemly linked tac promotors placed upstream of a full-length cDNA. By selecting Escherichia coli strain KY1436 as the host micro-organism, we established an efficient system for the production of human liver arginase protein. Chromatographies on CM-Sephadex G-150, DEAE-cellulose and Sephadex G-150, followed by preparative agar-gel electrophoresis, yielded 10 mg of apparently homogeneous enzyme protein from 1 g (wet wt.) of E. coli cells. E. coli-expressed human liver arginase had chemical, immunological and most catalytic properties indistinguishable from those of purified human erythrocyte arginase. However, E. coli-expressed arginase was a monomer of Mr 35,000, whereas the purified erythrocyte arginase was trimer of Mr 105,000. They differed also in pH- and temperature-stabilities. Gel-filtration experiments with these two purified arginases under various conditions, as well as with unfractionated human liver and erythrocyte cytosol preparations, indicated that the native form of human arginase should be of Mr 35,000, and that the trimeric appearance of human erythrocyte arginase after purification was an artifact of the purification procedures. It was thus concluded that, in Nature, the liver and erythrocyte arginases are identical proteins.  相似文献   

6.
7.
The present study investigated the ability of the arginine analog L-NAME (N(omega)-Nitro-L-arginine methyl ester) to modulate the activity of arginase. L-NAME inhibited the activity of arginase in lysates from rat colon cancer cells and liver. It also inhibited the arginase activity of tumor cells in culture. Furthermore, in vivo treatment of rats with L-NAME inhibited arginase activity in tumor nodules and liver, and the effect persisted after treatment ceased. The effect of L-NAME on arginase requires consideration when it is used in vivo in animal models with the aim of inhibiting endothelial NO-synthase, another enzyme using arginine as substrate.  相似文献   

8.
The present study investigated the ability of the arginine analog L -NAME (Nω-Nitro- L -arginine methyl ester) to modulate the activity of arginase. L -NAME inhibited the activity of arginase in lysates from rat colon cancer cells and liver. It also inhibited the arginase activity of tumor cells in culture. Furthermore, in vivo treatment of rats with L -NAME inhibited arginase activity in tumor nodules and liver, and the effect persisted after treatment ceased. The effect of L -NAME on arginase requires consideration when it is used in vivo in animal models with the aim of inhibiting endothelial NO-synthase, another enzyme using arginine as substrate.  相似文献   

9.
Liver cells isolated from the adult rat livers under mild conditions were preincubated for 1 day with Williams medium E (WE) containing serum, dexamethasone and insulin, and then the cells (monolayered) were incubated for 2-3 days with WE (1 ml) containing only insulin to measure DNA synthesis and/or mitosis. DNA synthesis of cultured liver cells was dependent on cell densities within a region from 0.1 X 10(6) to 1.0 X 10(6) nuclei/dish (Falcon, diameter 35 mm). The addition of EGF from the beginning of preincubation stimulated DNA synthesis (or replication) as well as cell proliferation in vitro, but the density-dependent inhibition of DNA synthesis was observed similarly in the presence of EGF. In contrast to the low and high density cultures, DNA synthesis in the intermediary density cultures was enhanced by enlarging the medium volume or by adding ornithine (arginase inhibitor). DNA synthesis in low density cultures was inhibited by liver plasma membranes in a concentration-dependent fashion. The inhibition of DNA synthesis by liver plasma membranes in low concentrations (less than 30 micrograms protein/ml) was reduced by adding either extra arginine or ornithine. DNA synthesis of cultured liver cells (low density) was inhibited by replacing arginine in WE with equimolar ornithine and urea or by adding a commercial arginase (bovine liver). These, together with earlier findings indicating the presence of arginase in liver plasma membranes (outer leaflet), seem to support the idea that arginase may be involved in density-dependent as well as plasma membrane-mediated inhibition of DNA synthesis of cultured liver cells. However, this does not exclude possible involvement of other inhibitory principle(s), such as direct cell-to-cell or cell-to-plasma membrane interactions, especially in higher cell densities or larger plasma membrane concentrations.  相似文献   

10.
Molecular characteristics of chicken liver arginase   总被引:2,自引:2,他引:0       下载免费PDF全文
A purification procedure for the preparation of chicken liver arginase in a homogeneous form is presented. The enzyme hydrolyses both arginine and argininic acid. Kinetic analysis reveals that the enzyme binds arginine when the amino group is protonated or unprotonated; however, the unprotonated form seems to be hydrolysed more rapidly. The enzyme exchanges with the medium approx. 1.6Mn(2+) ions per molecule.  相似文献   

11.
An arginase isolated from a capsulated Bacillus anthracis strain was highly purified and crystallized. The chemical and immunological characteristics of this enzyme re described. Some very important properties differ from those of another bacterial arginase, i.e. Staphylococcus aureus arginase, described in a previous paper (Soru et al. (2)). The two arginases have different crystallization forms, different molecular weight, Km, thermostability, Arrhenius activation energy. They have another N-terminal group and are immunologically strictly specific. These differences point to distinct proteins. The fact that two arginases of different origin are structurally non-identical suggests that they may be involved in different metabolic processes. Staphylococcal arginase was shown to participate in a complete ureogenetic cycle, for it also possesses the other enzymes of the cycle (Soru et al. (2)). Except arginase, no other enzyme of this cycle was identified in the capsulated B. anthracis strain. Arginase may be involved in another metabolic pathway, one that is important for the strain, such as the synthesis of glutamic acid, since the capsular material of the strain is a polymer gamma-linked polyglutamic acid, mainly configuration D (Ivanovic and Bruckner (20)). The fact that the N-terminal residue of B. anthracis arginase is a tetramer containing glutamic acid together with proline (in addition to alanine and glycine) suggests that arginase may participate as a regulatory enzyme in the synthesis of glutamic acid from proline via ornithine and arginine, respectively. This pathway is found in many bacteria. The proline oxidase system, which is supposed to catalyse the conversion of proline to glutamic acid, is under study now in Bacillus anthracis strains.  相似文献   

12.
After seed germination, hydrolysis of storage proteins provides a nitrogen source for the developing seedling. In conifers the majority of these reserves are located in the living haploid megagametophyte tissue. In the developing loblolly pine (Pinus taeda L.) seedling an influx of free amino acids from the megagametophyte accompanies germination and early seedling growth. The major component of this amino acid pool is arginine, which is transported rapidly and efficiently to the seedling without prior conversion. This arginine accounts for nearly half of the total nitrogen entering the cotyledons and is likely a defining factor in early seedling nitrogen metabolism. In the seedling, the enzyme arginase is responsible for liberating nitrogen, in the form of ornithine and urea, from free arginine supplied by the megagametophyte. In this report we investigate how the seedling uses arginase to cope with the large arginine influx. As part of this work we have cloned an arginase cDNA from a loblolly pine expression library. Analysis of enzyme activity data, accumulation of arginase protein and mRNA abundance indicates that increased arginase activity after seed germination is due to de novo synthesis of the enzyme. Our results suggest that arginase is primarily regulated at the RNA level during loblolly pine seed germination and post-germinative growth.  相似文献   

13.
Todd CD  Gifford DJ 《Planta》2003,217(4):610-615
Following germination of loblolly pine (Pinus taeda L.) seeds, storage proteins in the embryo and megagametophyte are broken down to provide nitrogen, in the form of amino acids, to the developing seedling. A substantial portion of the free amino acids released in this process is arginine. Arginine is hydrolyzed in the cotyledons of the seedling by the enzyme arginase (EC 3.5.3.1), which is under developmental control. It has been shown previously that the seedling is able to initiate arginase gene expression in vitro in the absence of the megagametophyte, however, presence of the megagametophyte causes a greater accumulation of arginase protein and mRNA. Using an in vitro culture system we show that arginine itself may be responsible for up-regulating arginase activity. Application of exogenous arginine to cotyledons of seedlings germinated in the absence of the megagametophyte caused an increase in total shoot pole arginase activity as well as arginase specific activity. Arginine was also able to induce arginase mRNA accumulation in the same tissue.  相似文献   

14.
Metabolite Alterations in the Genetically Spastic Mouse   总被引:1,自引:0,他引:1  
Abstract: The concentration of soluble amino acids in the brains of homozygous spastic mice was measured. It was found that brain arginine levels were reduced by 3% and urea concentration was 50% higher in affected mice. The activity of the brain and liver enzymes for arginine synthesis were similar in spastics and in controls. However, in spastic mice the activity of brain arginase is elevated. This increased arginase activity accounts for the decreased arginine concentration in the mutant mice.  相似文献   

15.
Summary The ornithine transaminase (EC.2.6.1.13) of Saccharomyces cerevisiae is induced by arginine, ornithine, and their analogs. Genetic regulatory elements which are involved in this induction process have been defined due to the isolation of specific mutants. Two classes of OTAse operator mutants have previously been described; three unlinked genes are presumed to code for a specific repressor, CARGR of both of the arginine catabolic enzymes, arginase, and ornithine transaminase. The level of transaminase of cells grown on ammonia plus arginine is much lower than it is when arginine is the sole nitrogen source. Ammonia thus seems to limit the amount of enzyme synthesized when arginine is present in the growth medium. Nevertheless, all attempts to disclose a nitrogen catabolite repression process in OTAse synthesis have failed; neither the action of mutations that release this regulation on arginase and other catabolic enzymes, nor the use of derepressing growth conditions, affect OTAse synthesis. A decrease of the cells' arginine pool when amonia or aminoacids (serine, glutamate) are added to arginine as a nitrogen nutrient results in a progressive reduction of transaminase synthesis. This suggests that arginine is the only physiological effector in those conditions: ammonia or some aminoacids would reduce the enzyme synthesis because of an inducer exclusion. The first stage of OTAse induction would then be operated by the CARGR repressor, and an additional regulatory element might take part in the full scale process. Preliminary data favoring the involvment of such an element are presented.  相似文献   

16.
Nitrogen regulation of arginase in Neurospora crassa.   总被引:5,自引:3,他引:2       下载免费PDF全文
The final products of the arginine catabolism that can be utilized as a nitrogen source in Neurospora crassa are ammonium, glutamic acid, and glutamine. The effect of these compounds on arginase induction by arginine was studied. In wild-type strain 74-A, induction by arginine was almost completely repressed by glutamic acid plus ammonium, whereas ammonium or glutamic acid alone had only moderate effects. Arginine products of catabolism also repressed arginase induction. A mutant, ure-1, which lacks urease activity, hyperinduced its arginase with arginine as a nitrogen source. The addition of either ammonium or glutamine produced effects similar to those in the wild-type strain. The effect of ammonium on arginase induction is mediated through its conversion into glutamine. This was demonstrated in mutant am-1, which lacks L-glutamate dehydrogenase activity. In this mutant, the effect of glutamic acid was reduced, and, with ammonium, it was completely lost. The addition of glutamine or glutamic acid plus ammonium to this strain decreased by threefold the induction of arginase by arginine. Proline, a final product of arginine catabolism, competitively inhibited arginase activity. This effect and the repression of arginase by glutamine are examples of negative modulation of the first enzyme in a catabolic pathway by its final products.  相似文献   

17.
18.
Liver ischemia-reperfusion (I/R) injury is associated with profound arginine depletion due to arginase release from injured hepatocytes. The purpose of this study was to determine whether arginase inhibition with N(omega)-hydroxy-nor-l-arginine (nor-NOHA) would increase circulating arginine levels and decrease hepatic damage during liver I/R injury. The effects of nor-NOHA were initially tested in normal animals to determine in vivo toxicity. In the second series of experiments, orthotopic syngeneic liver transplantation (OLT) was performed after 18 h of cold ischemia time in Lewis rats. Animals were given nor-NOHA (100 mg/kg) or saline before and after graft reperfusion. In normal animals treated with nor-NOHA, there were no histopathological changes to organs, liver enzymes, serum creatinine, or body weight. In the OLT model, animals treated with saline exhibited markedly elevated serum transaminases and circulating arginase protein levels. Nor-NOHA administration blunted the increase in serum arginase activity by 80% and preserved serum arginine levels at 3 h after OLT. Nor-NOHA treatment reduced post-OLT serum liver enzyme release by 50%. Liver histology (degree of necrosis) in nor-NOHA-treated animals was markedly improved compared with the saline-treated group. Furthermore, use of the arginase inhibitor nor-NOHA did not influence polyamine synthesis owing to the decrease in ornithine levels. Arginase blockade represents a potentially novel strategy to combat hepatic I/R injury associated with liver transplantation.  相似文献   

19.
Assay and kinetics of arginase   总被引:1,自引:0,他引:1  
A sensitive colorimetric assay for arginase was developed. Urea produced by arginase was hydrolyzed to ammonia by urease, the ammonia was converted to indophenol, and the absorbance was measured at 570 nm. The assay is useful with low concentrations of arginase (0.5 munit or less than 1 ng rat liver arginase) and with a wide range of arginine concentrations (50 microM to 12.5 mM). Michaelis-Menten kinetics and a Km for arginine of 1.7 mM were obtained for Mn2+-activated rat liver arginase; the unactivated enzyme did not display linear behavior on double-reciprocal plots. The kinetic data for unactivated arginase indicated either negative cooperativity or two types of active sites on the arginase tetramer with different affinities for arginine. The new assay is particularly well suited for kinetic studies of activated and unactivated arginase.  相似文献   

20.
Because arginase hydrolyzes arginine to produce ornithine and urea, it has the potential to regulate nitric oxide (NO) and polyamine synthesis. We tested whether expression of the cytosolic isoform of arginase (arginase I) was limiting for NO or polyamine production by activated RAW 264.7 macrophage cells. RAW 264.7 cells, stably transfected to overexpress arginase I or beta-galactosidase, were treated with interferon-gamma to induce type 2 NO synthase or with lipopolysaccharide or 8-bromo-cAMP (8-BrcAMP) to induce ornithine decarboxylase. Overexpression of arginase I had no effect on NO synthesis. In contrast, cells overexpressing arginase I produced twice as much putrescine after activation than did cells expressing beta-galactosidase. Cells overexpressing arginase I also produced more spermidine after treatment with 8-BrcAMP than did cells expressing beta-galactosidase. Thus endogenous levels of arginase I are limiting for polyamine synthesis, but not for NO synthesis, by activated macrophage cells. This study also demonstrates that it is possible to alter arginase I levels sufficiently to affect polyamine synthesis without affecting induced NO synthesis.  相似文献   

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