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PubMed2005

Dynamical flexibility and proton transfer in the arginase active site probed by ab initio molecular dynamics.

We have used ab initio molecular dynamics (AIMD) to investigate the dynamical flexibility of the bridged binuclear structural motif in the active site of arginase. Dynamical transformations play a crucial role in catalysis. We have provided direct insight into the motions of the first-shell ligands with emphasis on the chelating and bridging carboxylates. In the case of the terminal Asp234 residue we observe changes in the binding mode (carboxylate shifts). AIMD dynamics of sufficient duration has allowed us to observe proton transfer from the bridging nucleophile to the catalytically essential Asp 128 residue and to map the underlying free energy surface in terms of simple reaction coordinates, such as the oxygen-oxygen distance Ro-o and the asymmetric stretch delta. This has provided valuable insight into the nature of the last step of the catalytic cycle. In addition, constrained molecular dynamics permitted us to compare the deprotonation free energy of the bridging nucleophile in the case of native versus metal-depleted arginase.

Arginase↗
PubMed1976

Arginase from human full-term placenta.

Arginase was purified about 1800-fold from extracts of human full-term placenta; the enzyme appeared to be homogenous by disc electrophoresis and molecular-sieve chromatography. The mol. wt. determination by gel filtration and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis yielded a value of 70000 for the most pure and the partially purified enzyme. The human placenta arginase is a metalloenzyme with an optimum pH of 9.1. The Km for L-arginine is 27 mM. L-Ornithine and L-lysine show competitive inhibition with Ki values of 6.3 and 14 mM respectively.

Arginase↗
PubMed1984

Arginase activity during pregnancy and lactation.

Arginase activity in liver, kidney, small intestine and stomach of pregnant and lactating rats as well as their plasma levels have been measured. Late pregnancy as well as weaning induced a parallel increase in enzyme activity that is not reflected in the circulating urea levels. The arginase activity changes have been related to known changes in feeding and nitrogen handling patterns as well as to hormonal variations.

Animals↗
PubMed2004

Arginase plays a pivotal role in polyamine precursor metabolism in Leishmania. Characterization of gene deletion mutants.

The polyamine pathway of protozoan parasites has been successfully targeted in anti-parasitic therapies and is significantly different from that of the mammalian host. To gain knowledge into the metabolic routes by which parasites synthesize polyamines and their precursors, the arginase gene was cloned from Leishmania mexicana, and Deltaarg null mutants were created by double targeted gene replacement and characterized. The ARG sequence exhibited significant homology to ARG proteins from other organisms and predicted a peroxisomal targeting signal (PTS-1) that steers proteins to the glycosome, an organelle unique to Leishmania and related parasites. ARG was subsequently demonstrated to be present in the glycosome, whereas the polyamine biosynthetic enzymes, in contrast, were shown to be cytosolic. The Deltaarg knockouts expressed no ARG activity, lacked an intracellular ornithine pool, and were auxotrophic for ornithine or polyamines. The ability of the Deltaarg null mutants to proliferate could be restored by pharmacological supplementation, either with low putrescine or high ornithine or spermidine concentrations, or by complementation with an arginase episome. Transfection of an arg construct lacking the PTS-1 directed the synthesis of an arg that mislocalized to the cytosol and notably also complemented the genetic lesion and restored polyamine prototrophy to the Deltaarg parasites. This molecular, biochemical, and genetic dissection of ARG function in L. mexicana promastigotes establishes: (i) that the enzyme is essential for parasite viability; (ii) that Leishmania, unlike mammalian cells, expresses only one ARG activity; (iii) that the sole vital function of ARG is to provide polyamine precursors for the parasite; and (iv) that ARG is present in the glycosome, but this subcellular milieu is not essential for its role in polyamine biosynthesis.

Amino Acid Sequence↗
PubMed2006

The arginase II gene is an anti-inflammatory target of liver X receptor in macrophages.

The liver X receptors (LXRs) are ligand-dependent transcription factors that have been implicated in lipid metabolism and inflammation. LXRs also inhibit the expression of inflammatory genes in macrophages, including inducible nitric oxide synthase (iNOS). Some of these actions are mediated through LXR antagonism of NF-kappaB activity. The potential for LXRs to positively regulate the expression of anti-inflammatory molecules, however, has not been explored. Here we show that the arginase II (ArgII) gene is a direct target for LXR regulation. ArgII catalyzes the conversion of L-arginine into L-ornithine and urea, leading to the synthesis of polyamines. Expression of ArgII is induced by LXR agonists in macrophage cell lines and primary murine macrophages in a receptor-dependent manner. The ArgII promoter contains a functional LXR response elements that mediates promoter induction by LXR/RXR (retinoid X receptor) in transfection assays. Since ArgII and iNOS utilize a common substrate, induction of ArgII expression has the potential to exert anti-inflammatory effects by shifting arginine metabolism toward polyamine synthesis at the expense of NO production. In support of this hypothesis, we demonstrate that forced expression of ArgII mimics the inhibitory effect of LXR activation on macrophage NO production. Furthermore, inhibition of arginase activity partially reverses the inhibitory effect of LXR agonists on NO production. These studies suggest that regulation of ArgII may contribute to the immunomodulatory effects of LXRs.

Animals↗
PubMed2002

Overexpression of arginase I in enterocytes of transgenic mice elicits a selective arginine deficiency and affects skin, muscle, and lymphoid development.

BACKGROUND: Arginine is required for the detoxification of ammonia and the synthesis of proteins, nitric oxide, agmatine, creatine, and polyamines, and it may promote lymphocyte function. In suckling mammals, arginine is synthesized in the enterocytes of the small intestine, but this capacity is lost after weaning. OBJECTIVE: We investigated the significance of intestinal arginine production for neonatal development in a murine model of chronic arginine deficiency. DESIGN: Two lines of transgenic mice that express different levels of arginase I in their enterocytes were analyzed. RESULTS: Both lines suffer from a selective but quantitatively different reduction in circulating arginine concentration. The degree of arginine deficiency correlated with the degree of retardation of hair and muscle growth and with the development of the lymphoid tissue, in particular Peyer's patches. Expression of arginase in all enterocytes was necessary to elicit this phenotype. Phenotypic abnormalities were reversed by daily injections of arginine but not of creatine. The expression level of the very arginine-rich skin protein trichohyalin was not affected in transgenic mice. Finally, nitric oxide synthase-deficient mice did not show any of the features of arginine deficiency. CONCLUSIONS: Enterocytes are important for maintaining arginine homeostasis in neonatal mice. Graded arginine deficiency causes graded impairment of skin, muscle, and lymphoid development. The effects of arginine deficiency are not mediated by impaired synthesis of creatine or by incomplete charging of arginyl-transfer RNA.

Animals↗
PubMed2003

D-arginase of Arthrobacter sp. KUJ 8602: characterization and its identity with Zn(2+)-guanidinobutyrase.

D-Arginase activity was found in the cells of an isolate, Arthrobacter sp. KUJ 8602, grown in the L-arginine medium, and the enzyme was purified and characterized. Its molecular weight was estimated to be about 232,000 by gel filtration, and that of the subunit was approximately 40,000 by SDS-PAGE, suggesting that the enzyme is a homohexamer. The enzyme acted on not only D-arginine but also 4-guanidinobutyrate, 3-guanidinopropionate and even L-arginine. The V(max)/K(m) values for 4-guanidinobutyrate and D-arginine were determined to be 87 and 0.81 micro mol/min/mg/mM, respectively. Accordingly, the enzyme is regarded as a kind of guanidinobutyrase [EC 3.5.3.7]. The pH optima for 4-guanidinobutyrate and D-arginine were 9.0 and 9.5, respectively. The enzyme was inhibited competitively by 5-aminovalerate, and thiol carboxylates such as mercaptoacetate served as strong mixed-type inhibitors. The enzyme contained about 1 g-atom of firmly bound Zn(2+) per mol of subunit, and removal of the metal ions by incubation with 1,10-phenanthroline resulted in loss of activity. The inactivated enzyme was reactivated markedly by incubation with either Zn(2+) or Co(2+), and slightly by incubation with Mn(2+). The nucleotide sequence of enzyme contains an open reading frame that encodes a polypeptide of 353 amino acid residues (M(r): 37,933). The predicted amino acid sequence contains sequences involved in the binding of metal ions and the guanidino group of the substrate, which show a high homology with corresponding sequences of Mn(2+)-dependent amidinohydrolases such as agmatinase from Escherichia coli and L-arginase from rat liver, though the homology of their entire sequences is relatively low (24-43%).

Animals↗
PubMed1994

L-canavanine influences feed intake, plasma basic amino acid concentrations and kidney arginase activity in chicks.

L-Canavanine [2-amino-4-(guanidinooxy) butyric acid], a non-protein amino acid that is structurally analogous to arginine, has been proposed as a major antinutritional factor responsible for the toxic effects induced by raw Canavalia ensiformis (L.) seeds in chicks. We investigated the effects of L-canavanine on performance and select metabolic responses of growing chicks. Canavanine was added to a control diet, in an amount equivalent to that provided by 300 g raw canavalia seeds/kg diet (10 g free base canavanine/kg diet). Growth, plasma basic amino acids and kidney arginase, activity were measured. The incorporation of canavanine into a nutritionally balanced diet for growing chicks depressed feed intake and growth by approximately 25% (P < 0.01) compared with the control diet. Performance was unaffected by equimolar amounts of arginine. Canavanine exerted its growth-depressing effect exclusively by reducing feed intake, because this effect was not observed in a pair-feeding experiment. Chicks fed a diet containing 473 mmol canavanine sulfate/kg for 11 d were given an intracrop dose of 946 mmol of canavanine sulfate or arginine hydrochloride. In both cases, plasma histidine and lysine concentrations were significantly decreased compared with a placebo group dosed with water. Plasma arginine concentration was unaffected by the canavanine sulfate dose but, as expected, was significantly increased by the arginine hydrochloride dose. Free base canavanine significantly (P < 0.05) reduced kidney arginase activity. No overt toxic effects were observed at any point during the study. These data indicate that, although canavanine is not the principal antinutritional factor in Canavalia ensiformis seeds, its presence in the diet precludes optimum performance of chicks.

Administration, Oral↗
PubMed1994

The delayed glucocorticoid-responsive and hepatoma cell-selective enhancer of the rat arginase gene is located around intron 7.

Liver-selective transcription of the gene for rat arginase, an ornithine cycle (urea cycle) enzyme, is induced by glucocorticoids in a delayed secondary manner; the mRNA induction by the hormones requires de novo protein synthesis, and is preceded by a time lag of several hours. We searched for a DNA element mediating the glucocorticoid induction of the arginase gene with a transient transfection system using hepatoma cell lines. Within the 233-base pair region that is located 11 kilobases downstream from the transcription start site and that spans the junction of intron 7 and exon 8, we detected an enhancer element that is glucocorticoid-responsive and hepatoma cell-selective. The time course of the glucocorticoid induction through this enhancer element was delayed compared to that through the primary glucocorticoid-responsive mouse mammary tumor virus promoter. Footprint analysis revealed four protein-binding sites in this enhancer region. In gel retardation analysis, each site exhibited a complicated profile characterized by a number of shifted bands, some of which were tissue-selective and others ubiquitous. Gel shift competition and antibody supershift/inhibition analysis demonstrated that two of the four sites are recognized by members of the CCAAT/enhancer binding protein (C/EBP) family, some of which are liver-enriched.

Animals↗
PubMed1976

Purification and properties of rat small intestinal arginase.

Arginase [L-arginine amidinhydrolase EC 3.5.3.1] from rat small intestine was purified about 2,200-fold and its properties were compared with those of the rat liver and kidney enzymes. Intestinal arginase was extremely labile on storage either at -10 degrees or 4 degrees and lost activity during purification unless 25 mM L-valine was present. The purified enzyme appeared to be homogeneous by disc electrophoresis and its molecular weight was estimated to be 120,000 by Sephadex G-100 filtration...

Amino Acids↗
PubMed2003

Divergent effects of GM-CSF and TGFbeta1 on bone marrow-derived macrophage arginase-1 activity, MCP-1 expression, and matrix metalloproteinase-12: a potential role during arteriogenesis.

Granulocyte/macrophage-colony stimulating factor (GM-CSF) and transforming growth factor (TGF)beta1 induce arteriogenesis in a nonischemic model of femoral artery ligation. Moreover, clinical trials demonstrated an improved collateralization after injection of bone marrow cells. In the present study, the expression of arteriogenic factors in bone marrow-derived macrophages (BMDM) was measured to verify the potential of these cells to influence collateral artery growth. GM-CSF induced in BMDM the expression of monocyte chemoattractive protein (MCP)-1, matrix-metalloproteinase (MMP)-12, and arginase-1-the latter also showing a remarkable increase in activity. During in vivo induced arteriogenesis, the accumulation rate of macrophages around proliferating collaterals was significantly increased. We also show that MCP-1 is found to be mainly expressed in the media of the vessel wall, MMP-12 in macrophages of the adventitia, and arginase at both locations. This study provides for the first time a comprehensive analysis of GM-CSF/TGFbeta1-regulated arteriogenic factors in BMDM and supports the hypothesis that arteriogenesis is a multistage mechanism, including monocyte/macrophage adhesion and transmigration, pro-arteriogenic cytokine expression, degradation of connective tissue, and collagen synthesis regulation. Selective modulation of these mechanisms as well as cell-based therapies supplying arteriogenic factors in vivo point toward new strategies to influence collateral artery growth.

Animals↗
PubMedDate not supplied

Consumption of L-arginine mediated by Entamoeba histolytica L-arginase (EhArg) inhibits amoebicidal activity and nitric oxide production by activated macrophages.

In this study we discuss the cloning and expression of Entamoeba histolytica arginase (EhArg), an enzyme that catalyses the hydrolysis of L-arginine to L-ornithine and urea. L-norvaline, a competitive inhibitor of E. histolytica L-arginase, inhibits the growth of the parasite, which suggests that the catabolism of L-arginine mediated by EhArg is essential. Nitric oxide (NO) is an antimicrobial agent that inhibits some key enzymes in the metabolism of Entamoeba histolytica. NO is synthesized by activated macrophages from L-arginine, the substrate of NO synthase (NOS-II). We show that E. histolytica inhibits NO mediated amoebicidal activity of activated macrophages by consuming L-arginine present in the medium.

Amino Acid Sequence↗
PubMed1990

Inhibitory effect of mycoplasma-released arginase. Activity in mixed-lymphocyte and tumour cell cultures.

Non-fermenting mycoplasma species deplete culture media for arginine through arginase activity linked to their arginine deiminase pathway, resulting in proliferation arrest and cell death in mycoplasma-contaminated cell cultures. The presence of only 2-3 Mycoplasma (M.) arginini-contaminated T cells in a one-way allogeneic mixed-lymphocyte culture (MLC) significantly inhibits development of cytotoxic T-cell activity. Likewise, strong degrees of inhibition are observed after addition of nanogram doses of M. arginini extracts (MAE) to MLC or cell proliferation cultures. M. arginini-induced cell inhibition can be reversed by addition of excess arginine to the culture medium. Antisera raised against non-fermenting, but not against fermenting, mycoplasma species block the inhibitory effect of MAE. SDS-PAGE separation of MAE disclosed a broad band at 60 kDa which contained arginase activity when assayed in MLC and cell proliferation culture. SDS-PAGE followed by western blotting and reaction with antisera raised against non-fermenting mycoplasma species demonstrated a band at 43 kDa common for these micro-organisms.

Animals↗
PubMed1978

Arginase deficiency in multiple tissues in argininemia.

A 5 9/12-year-old Mexican female with argininemia presented at 4 years of age with severe growth retardation, microcephaly, mental retardation, loss of ability to walk, spasticity and epileptiform electroencephalogram. At follow-up, blood ammonia was elevated only twice out of 30 determinations. Blood arginine was 544 to 1,074 mumol/l (normal 61 to 173); cerebrospinal fluid arginine was 88 mumol/l (normal 6 to 29); and urinary arginine, citruline and argininosuccinic acid were consistently elevated. Arginase activities in tissues from the propositus were 0.01 mU/mg hemoglobin in erythrocytes (normal 29.8 to 96.1); 9 mU/mg protein in liver (normal 1,522 to 5,491); and 5 mU/mg protein in stratum corneum (normal 2,856 to 7,556). The demonstration of arginase deficiency in liver and stratum corneum suggests a generalized deficiency and helps to explain the elevation of blood arginine. Therapeutic trials of orally administered lysine to enhance dibasic amino acid competition and of enzyme replacement using erythrocyte transfusion did not result in significantly decreased blood arginine or clinical improvement.

Abnormalities, Multiple↗
PubMed1983

Spectroscopic study on the structure and stability of beef liver arginase.

The secondary and tertiary structure of the oligomeric arginase (EC 3.5.3.1) from beef liver was investigated by circular dichroism (CD) and fluorescence measurements. The far-ultraviolet CD spectrum of the enzyme at neutral pH is indicative of high helical content. The intrinsic fluorescence emission of the protein is due to tryptophan, the contribution of tyrosine being small. Upon excitation at 295 nm, the maximum of emission occurs at 330 nm, implying that the tryptophan residues are rather buried in a hydrophobic interior of the protein. Ethylenediaminetetraacetic acid (EDTA), which inactivates the enzyme by removing the functional Mn2+-ion from the enzyme, does not dissociate the enzyme into subunits, nor affect noticeably its secondary and tertiary structure. Inactivation occurs in the acid pH range, being complete at pH below 4. However, acidification up to pH 1.5 produced only limited changes in the far-ultra-violet CD spectrum and intrinsic fluorescence emission properties. The enzyme shows noteworthy thermal stability, as shown by measuring the residual activity after heating and by evaluating the temperature dependence of the CD signal at 220 nm and the intensity of emission fluorescence. A temperature of half inactivation (Tm) of 77 degrees was determined upon heating the enzyme at pH 7.5 in the presence of Mn2+-ions for 10 min; in the presence of EDTA, Tm is shifted to 55 degrees. Taken together, these observations indicate that the structural stability of beef liver arginase arises from a clustering of hydrophobic amino acids and from Mn2+-ion binding.

Animals↗
PubMed1975

Purification of arginases from human-leukemic lymphocytes and granulocytes: study of their physicochemical and kinetic properties.

Arginase has been isolated from granulocytes of a patient with chronic myelocytic leukemia and from lymphocytes of a patient with chronic lymphocytic leukemia and both enzymes have been purified to apparent homogeneity. The purification procedure employed acetone extraction, ammonium sulfate precipitation, DEAE-cellulose and CM-Sephadex chromatography and gel filtration on Bio-Gel A 1.5m. Both enzymes appear to be metalloenzymes, and to have molecular weights of about 120 000. Studies with the dissociated enzymes suggest that the subunit molecular weight is about 37 000, in agreement with a tetrameric aggregate structure of the native enzymes. Human leukemic granulocyte and lymphocyte arginases are strongly basic proteins with pI values between 9.25 and 9.35. Their free -SH groups enabled them to be linked to organomercurial-agarose. The kinetic properties estimated for both enzymes showed an optimum pH of 8.5, and an optimal MnCl2 concentration of 0.01 M. The Km for L-arginine is 2.7-3.1 mM and L-ornithine exhibits a mixed type of inhibition, with a Ki of 15.5-15.7 mM.

Arginase↗
PubMedDate not supplied

Amino acids, arginase and nitric oxide in vascular health.

1. Nitric oxide (NO) plays a fundamental role in the vasculature because of its diverse influence in vascular protection, including its well-reported antiproliferative, anti-inflammatory, antithrombotic and vasodilator effects. In many vascular disease states, NO production is reduced as a result of endothelial dysfunction, in part caused by a decrease in substrate (L-arginine) availability. 2. The role of L-arginine and other amino acids important in nitrogen balance has been re-examined in the context of their effects on vascular health. The metabolism of L-arginine is complex because it is involved in a plethora of other pathways, such as urea, creatine and agmatine production. L-Arginine supplementation in patients with vascular disease is well reported to benefit patients therapeutically because of its effect on both NO-dependent and -independent mechanisms. 3. L-Arginine availability depends on the flux of other amino acids in the body, including L-glutamine, L-glutamate, L-ornithine, L-citrulline and L-lysine. The role of L-methionine and homocystine and their effect on NO also play an influential role in the body. 4. Recent data suggest that the key enzyme involved in the L-arginine-urea cycle, arginase, is coexpressed in NO-producing cells in the vasculature. In the present review, we examine the potential role of arginase as a therapeutic target for vascular health.

Amino Acids↗