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Complex regulation of T cell functions during pregnancy is required to ensure materno-fetal tolerance. Here we reveal a novel pathway for the temporary suppression of maternal T cell responses in uncomplicated human pregnancies. Our results show that arginase activity is significantly increased in the peripheral blood of pregnant women and remarkably high arginase activities are expressed in term placentae. High enzymatic activity results in high turnover of its substrate L-arginine and concomitant reduction of this amino acid in the microenvironment. Amino acid deprivation is emerging as a regulatory pathway of lymphocyte responses and we assessed the consequences of this enhanced arginase activity on T cell responses. Arginase-mediated L-arginine depletion induces down-regulation of CD3 zeta, the main signalling chain of the TCR, and functional T cell hyporesponsiveness. Importantly, this arginase-mediated T cell suppression was reversible, as inhibition of arginase activity or addition of exogenous L-arginine restored CD3 zeta chain expression and T cell proliferation. Thus, L-arginine metabolism constitutes a novel physiological mechanism contributing to the temporary suppression of the maternal immune response during human pregnancy.

Arginase 1, an enzyme induced by Th2 cytokines, is a hallmark of alternatively activated macrophages and is responsible for the hydrolysis of L-arginine into ornithine, the building block for the production of polyamines. Upregulation of arginase 1 has been observed in a variety of diseases, but the mechanisms by which arginase contributes to pathology are not well understood. We reveal here a unique role for arginase 1 in the pathogenesis of nonhealing leishmaniasis, a prototype Th2 disease, and demonstrate that the activity of this enzyme promotes pathology and uncontrolled growth of Leishmania parasites in vivo. Inhibition of arginase activity during the course of infection has a clear therapeutic effect, as evidenced by markedly reduced pathology and efficient control of parasite replication. Despite the clear amelioration of the disease, this treatment does not alter the Th2 response. To address the underlying mechanisms, the arginase-induced L-arginine catabolism was investigated and the results demonstrate that arginase regulates parasite growth directly by affecting the polyamine synthesis in macrophages.

The balance of arginine metabolism via nitric oxide synthase (NOS) or arginase is an important determinant of the inflammatory response of murine macrophages and dendritic cells. Here we analyzed the expression of the isoform arginase I in human myeloid cells. Using healthy donors and patients with arginase I deficiency, we found that in human leukocytes arginase I is constitutively expressed only in granulocytes and is not modulated by a variety of proinflammatory and anti-inflammatory stimuli in vitro. We demonstrate that arginase I is localized in azurophil granules of neutrophils and constitutes a novel antimicrobial effector pathway, likely through arginine depletion in the phagolysosome. Our findings demonstrate important differences between murine and human leukocytes with respect to regulation and function of arginine metabolism via arginase.

Arginase activity was measured in serum and biopsy from healthy individuals and colorectal cancer patients. Arginase activity in tumor samples (87 +/- 7.7 U/g tissue) was significantly higher than in controls (40.7 +/- 3.3 U/g tissue). However, serum arginase activity did not show any significant change in both groups. Finally, the micromethod used to quantify arginase activity in this study is superior to other methods because it has increased sensitivity, requires less sample, and is less time-consuming. Arginase differences are significant, according to the t-test (P<0.05)

One determinant that could play a role in the quaternary structure of human arginase is the pair of salt links between the strictly conserved residues R255 from one monomer and E256 from every adjacent subunit. In this work, the ionic interaction between monomers was disrupted by expressing a human arginase where Glu-256 had been substituted by Gln. Biochemical analyses of the mutant protein showed that: (i) it shares the wild-type kinetic parameters of the arginine substrate; (ii) E256Q arginase behaves as a monomer by gel filtration; (iii) it is drastically inactivated by dialysis in the presence of EDTA, an inhibitory effect which is reversed by addition of Mn(2+); and (iv) the mutant enzyme loses thermal stability. The lack of oligomerisation for E256Q arginase and the conservation of E256 throughout evolution of the protein family suggest that this residue is involved in the quaternary structure of arginases.

Arginase I is a homotrimeric protein with a binuclear manganese cluster. At the C-terminus of each monomer, the polypeptide chain forms an unusual S-shaped oligomerization motif where the majority of intermonomer contacts are located [Z.F. Kanyo, L.R. Scolnick, D.E. Ash, D.W. Christianson, Nature 383 (1996) 554-557]. In order to study the implication of this motif in the quaternary structure of human arginase I, we have constructed a truncated arginase lacking the 14 C-terminal amino acids, leaving Arg-308 as the last residue in the sequence. The resulting protein retains its trimeric structure, as determined by gel filtration (molecular mass 94 kDa). The same result was obtained in the presence of high ionic strength (KCl 0.5 M). Both data indicate that neither the S-shaped motif nor Arg-308 are fundamental in keeping the trimeric quaternary structure. Data obtained from intrinsic anisotropy and fluorescence intensity studies allow us to predict that the distance between the two unique tryptophans in the sequence is 2.9 nm in the native arginase and 4.1 nm for the truncated mutant. These distances allow us to assume a different conformational state in the truncated arginase without any change in its quaternary structure, suggesting that the carboxy-terminal motif is not the most prominent domain implicated in the quaternary structure of human arginase. Collisional quenching studies reinforce this possibility, since using I(-) as quenching molecule we were able to distinguish the two tryptophans in the truncated arginase. Moreover, kinetic studies show that the truncated mutant was fully active. In summary, the main conclusion about the structure of the human arginase I, derived from our study, is that the C-terminal S-shaped motif is not basic to the maintenance of the quaternary structure nor to the activity of the protein.

Activated murine macrophages metabolize arginine by two alternative pathways involving the enzymes inducible NO synthase (iNOS) or arginase. The balance between the two enzymes is competitively regulated by Th1 and Th2 T helper cells via their secreted cytokines: Th1 cells induce iNOS, whereas Th2 cells induce arginase. Whereas the role of macrophages expressing iNOS as inflammatory cells is well established, the functional competence of macrophages expressing arginase remains a matter of speculation. Two isoforms of mammalian arginases exist, hepatic arginase I and extrahepatic arginase II. We investigated the regulation of arginase isoforms in murine bone marrow-derived macrophages (BMMPhi) in the context of Th1 and Th2 stimulation. Surprisingly, in the presence of either Th2 cytokines or Th2 cells, we observe a specific induction of the hepatic isoform arginase I in BMMPhi. Induction of arginase I was shown on the mRNA and protein levels and obeyed the recently demonstrated synergism among the Th2 cytokines IL-4 and IL-10. Arginase II was detectable in unstimulated BMMPhi and was not significantly modulated by Th1 or Th2 stimulation. Similar to murine BMMPhi, murine bone marrow-derived dendritic cells, as well as a dendritic cell line, up-regulated arginase I expression and arginase activity upon Th2 stimulation, whereas arginase II was never detected. In addition to revealing the unexpected expression of arginase I in the macrophage/monocyte lineage, these results uncover a further intriguing parallelism between iNOS and arginase: both have a constitutive and an inducible isoform, the latter regulated by the Th1/Th2 balance.

Arginase is induced in bone marrow-derived macrophages by agents that increase the intracellular concentrations of cAMP (Br-cAMP, prostaglandin E2) and, in their presence, the LPS induced NO synthesis is down regulated. Moreover, interleukin 10 which induces arginase in macrophages is able to increase the cAMP-dependent protein kinase A activity. In contrast, suppressors of NOS synthesis like protein kinase C inhibitors and calmodulin antagonists (W7), or NO activators (A23187) have no effect on the induction of arginase by LPS. These results strongly suggest that PKA is involved in the induction of arginase and supports the hypothesis that there is a reciprocal regulation of these two enzymes that drives the macrophages towards opposite functional states.

Activation with lipopolysaccharide induces macrophages to produce the enzymes arginase and nitric oxide (NO) synthase. Both enzymes use as a substrate the amino acid L-arginine, which can be either hydrolyzed by arginase to urea and ornithine or oxidized by NO synthase to NO and citrulline. NO is important in the bactericidal and cytotoxic activities of macrophages. An equivalent functional role of arginase and its products is not known. We tested the induction of arginase in bone marrow-derived macrophages by endogenous mediators that are known to induce NO synthase, such as interferon-gamma (IFN-gamma), or suppress the induction of this enzyme, such as interleukin (IL)-4, IL-10, and prostaglandin E2 (PGE2). We find that PGE2 and the TH2 cytokines IL-4 and IL-10 are potent inducers of arginase. In contrast, the TH1 cytokine IFN-gamma does not induce arginase. Simultaneous application of both types of mediators leads to reduced induction of both arginase and NO synthase. Exposure of macrophage cultures to inducers of NO synthase exhausts their ability to respond subsequently to inducers of arginase. Conversely, exposure of the cells to inducers of arginase exhausts their ability to respond subsequently to inducers of NO synthase. The results are consistent with a competition of both enzymes for their substrate, L-arginine, with a reciprocal inhibition in the induction of both enzymes, or a combination of both phenomena. The enzymes NO synthase and arginase appear to define two alternate functional states of macrophages, induced by TH1 and TH2 cytokines, respectively.

The present study addresses the regulatory mechanisms involved in the arginine metabolism of macrophages by arginase and nitric oxide synthase. Induction of both enzymes with LPS or by mixed lymphocyte reaction has been reported. Here, we demonstrate that these enzymes can be independently induced in murine bone-marrow-derived macrophages with the appropriate agonists. Arginase expression is specifically triggered by IL-4, IL-10, PGE2 as well as non-toxic or detoxified LPS. Conversely, IFN gamma induces only NO synthesis in these cells. The results demonstrate that the metabolism of arginine in macrophages is controlled by TH1/TH2-dependent cytokines and suggest a regulatory role of arginase on the NO synthesis by intracellular substrate depletion.

We propose a modification of Schimke's method for urea determination as a valuable micromethod for measuring arginase in activated macrophages. The method exhibits the following advantages: (a) it uses small amounts of samples (approximately 25,000 macrophages per assay); (b) it does not interfere with other related metabolites that are also present in the activated macrophage such as citrulline or arginine; (c) saturating concentrations of the substrate arginine can be used; and (d) it is much more sensitive than Schimke's method and can detect small amounts of urea, in the order of 0.02 mumol.

Some kinetic and regulatory properties of lactating rat mammary gland arginase were studied. At pH 7.4, i.e. at near-physiological conditions, there was evidence of inhibition by excess of substrate, with a Km value of 9.5 mM, slightly lower than the value of 18 mM observed at pH 9.8 (maximum enzyme activity). A study was also made of the effects of proline, ornithine, lysine and certain branched-chain aminoacids on enzyme activity: lactating rat mammary gland arginase was strongly and competitively inhibited by lysine, ornithine and valine, with Ki values of 1.2 mM, 1.1 mM and 3.6 mM, respectively. Other aminoacids (proline, isoleucine and leucine) also inhibited lactating rat mammary gland arginase, although to a lesser extent.

A detailed understanding of the regulatory mechanisms of arginase in the cell will depend on the clarification of the origin of the two different molecular mass subunits and on the arrangements of them to constitute the native enzyme. Here, we show the immunological recognition of the 39.5 and 37.0 kDa subunits of arginase by antibodies against both subunits. We also find that the subunit stoichiometry (39.5 kDa: 37.0 kDa) present in purified arginase preparations as well as in fresh isolated microsomes and cytoplasm corresponds to 3:1, indicating high prevalence of a constant arrangement of the constitutive subunits of arginase. These findings represent evidence for a limited posttranscriptional or posttranslational modification of only a fraction of the synthesized arginase in liver.

1. Rat mammary gland arginase is a metallo-enzyme dependent on Mn2+, which can be only partially substituted by Cd2+. 2. Reconstitution of the activity of dialyzed arginase by manganese is a two-phase process; the second phase is independent of the cation concentration, with a half-time recovery (t1/2) of 10.77 min. 3. The apparent Km for Mn2+ is 280 microM and 10.5 microM for enzyme dialyzed for 24 and 72 hr, respectively. 4. Treatment with 5 mM EDTA at pH 6 totally inhibits enzyme activity, which is reconstituted by Mn2+. 5. Results obtained with iodoacetamide treatment suggest the existence of sulphydryl groups accessible only when the enzyme is dialyzed.

1. Controlled tryptic digestion of native arginase from rat liver suggests that Mn2+ promotes a stable conformation as shown by the following features. 2. An 18-fold increase in the half-life of arginase activity in the presence of Mn2+ is produced. 3. The stability of subunit B of arginase is increased in the presence of Mn2+ as revealed by SDS-PAGE during the time-course of trypsin cleavage. 4. The different digestion products of arginase with and without Mn2+ appearing during the time-course of tryptic treatment. 5. Different activity/bands protein ratio at any time of the tryptic digestion in the incubation mixtures, with and without Mn2+, are apparent.

The phototrophic bacterium Rhodobacter capsulatus E1F1 grew with L-arginine or L-homoarginine as nitrogen source under light/anaerobiosis. However, when L-arginine was used as the only source of both carbon and nitrogen, the bacterium exhibited weak growth levels and the excess of nitrogen was excreted to the medium as ammonia. By contrast, L-ornithine was used under phototrophic conditions as either nitrogen or carbon source. Other compounds of the arginine catabolic pathways, such as putrescine or proline, also supported phototrophic growth of this bacterium. Under heterotrophic/dark conditions, R. capsulatus always showed a low growth rate with those nitrogen compounds. Cells growing on media containing L-arginine, L-homoarginine or L-ornithine induced an Mn(2+)-dependent arginase activity regardless of the presence of ammonium ions and other readily utilizable nitrogen sources. Arginase activity was strongly inhibited by Zn2+, Cu2+, borate, L-cysteine, L-ornithine and gamma-guanidinobutyrate. Mercurials also inactivated arginase, the activity being partially restored by the presence of thiols. Arginase was purified to electrophoretic homogeneity and found to consist of four identical subunits of 31 kDa. The molecular parameters and kinetic constants of arginase from R. capsulatus E1F1 resembled those previously described for the Saccharomyces cerevisiae enzyme rather than those of bacterial arginases.

Rat liver plasma-membrane-bound arginase was investigated in order to obtain data regarding its physico-chemical properties. Arginase bound to plasma membrane presented a specific activity of 0.74 +/- 0.09 IU/mg for the fully-activated enzyme, the pH of maximum activity being 9.8. Maximum stability was recorded at two pH values, 7 and 10.5 respectively. Mn2+ activated the enzyme, while Cu2+ and Zn2+, and to a lesser extend Co2+, showed a strong inhibitory effect. Ca2+ and Mg2+ had no effect at the concentrations assayed. The influence of temperature was studied in the presence and in the absence of Mn2+. The enzyme was stable up to 65 degrees C in both cases. Membrane- bound arginase showed an activation energy of 11.5 +/- 1.4 Kcal/mol between 20 and 40 degrees C, and 13.3 +/- 2.5 Kcal/mol between 40 and 60 degrees C. The Q10 for the same temperature ranges were 1.78 and 1.9 respectively. The membrane-bound enzyme presented two different Michaelis constants, one with high affinity (2.05 +/- 0.73 mM) and the other with low affinity for arginine (130 +/- 27.2 mM). Solubilized arginase showed very similar values. Among all the structural analogous assayed, only L-canavanine proved to be substrate for arginase, with and L-arginine/L-canavanine hydrolysis ratio of 5.8 +/- 0.28. No reactivity was found between plasma-membrane-bound arginase and anti-rat liver arginase antibodies raised in rabbits.

Rat liver arginase was covalently trapped in a fibrin clot. Among the physicochemical properties of the enzyme studied were Mn2+ requirement, pH behavior, temperature and time stability, effect of denaturing agents, and kinetic properties. The immobilized arginase showed the same substrate affinity as soluble arginase, but had higher stability at room temperature, was more resistant to denaturation, and had a higher catalytic activity at physiological pH. The properties so far examined may enhance the use of immobilized arginase in cancer therapy.

Arginase (E.C. 3.5.3.1) the enzyme which catalyses the hydrolysis of arginine to ornithine and urea has been investigated in guinea pig liver in relation to the kinetic constants of its substrate and inhibitors as well as to other physico-chemical properties. The results show that the enzyme has Km value of 19.6 mM for its substrate L-arginine and is competitively inhibited by one of its reaction products, the L-ornithine, and also by L-lysine and diaminopymelic acid. Optimal activity of the enzyme occurs at 10.5 pH and maximal stability in the range of 6.5 to 7.5 pH. The mentioned arginase exhibits temperature dependent activity and stability, being 64 degrees C (15 min and pH 7.5) the half-inactivation temperature. An increase in the activity and temperature stability of the enzyme, when previously activated by heating for 5 min at 45 degrees C in the presence of 10 mM MnCl2, has been achieved.

The circadian rhythms of glucose 6-phosphate dehydrogenase (G6PD) and of arginase activities and total protein content have been studied in the livers of 24-h fasted rats. Both G6PD and arginase activities reach a maximum at night and a minimum during the light period. On the other hand, the total protein level was maximal during the light period while it decreased to its lowest level during the dark period. These results are in agreement with the existence of a lipogenesis-lipolysis circadian rhythm in the rat, since the higher G6PD activity at night provides the necessary NADPH for lipid biosynthesis. The increase in arginase activity is also in agreement with an increase in amino-acid catabolism, probably as a source of energy and metabolic intermediates.

The influence of fasting and cortisol treatment on the levels of arginosuccinate synthetase (E.C. 6.3.4.5.), ARGINOSUCCINASe (E.C. 4.3.2.1) and arginase (C.E. 3.5.3.1) from guinea pig liver has been investigated. The results show a general increase in the levels of the mentioned enzymes, with specific enzyme synthesis for fasting, and unspecific for cortisol treatment.