L-Ornithine Derived Polyamines in Cystic Fibrosis Airways

Increased arginase activity contributes to airway nitric oxide (NO) deficiency in cystic fibrosis (CF). Whether down-stream products of arginase activity contribute to CF lung disease is currently unknown. The objective of this study was to test whether L-ornithine derived polyamines are present in CF airways and contribute to airway pathophysiology. Polyamine concentrations were measured in sputum of patients with CF and in healthy controls, using liquid chromatography-tandem mass spectrometry. The effect of spermine on airway smooth muscle mechanical properties was assessed in bronchial segments of murine airways, using a wire myograph. Sputum polyamine concentrations in stable CF patients were similar to healthy controls for putrescine and spermidine but significantly higher for spermine. Pulmonary exacerbations were associated with an increase in sputum and spermine levels. Treatment for pulmonary exacerbations resulted in decreases in arginase activity, L-ornithine and spermine concentrations in sputum. The changes in sputum spermine with treatment correlated significantly with changes in L-ornithine but not with sputum inflammatory markers. Incubation of mouse bronchi with spermine resulted in an increase in acetylcholine-induced force and significantly reduced nitric oxide-induced bronchial relaxation. The polyamine spermine is increased in CF airways. Spermine contributes to airways obstruction by reducing the NO-mediated smooth muscle relaxation.     

Crystal structures of Bacillus caldovelox arginase in complex with substrate and inhibitors reveal new insights into activation, inhibition and catalysis in the arginase superfamily

BACKGROUND: Arginase is a manganese-dependent enzyme that catalyzes the hydrolysis of L-arginine to L-ornithine and urea. In ureotelic animals arginase is the final enzyme of the urea cycle, but in many species it has a wider role controlling the use of arginine for other metabolic purposes, including the production of creatine, polyamines, proline and nitric oxide. Arginase activity is regulated by various small molecules, including the product L-ornithine. The aim of these structural studies was to test aspects of the catalytic mechanism and to investigate the structural basis of arginase inhibition. RESULTS: We report here the crystal structures of arginase from Bacillus caldovelox at pH 5.6 and pH 8.5, and of binary complexes of the enzyme with L-arginine, L-ornithine and L-lysine at pH 8.5. The arginase monomer comprises a single compact alpha/beta domain that further associates into a hexameric quaternary structure. The binary complexes reveal a common mode of ligand binding, which places the substrate adjacent to the dimanganese centre. We also observe a conformational change that impacts on the active site and is coupled with the occupancy of an external site by guanidine or arginine. CONCLUSIONS: The structures reported here clarify aspects of the active site and indicate key features of the catalytic mechanism, including substrate coordination to one of the manganese ions and an orientational role for a neighboring histidine residue. Stereospecificity for L-amino acids is found to depend on their precise recognition at the active-site rim. Identification of a second arginine-binding site, remote from the active site, and associated conformational changes lead us to propose a regulatory role for this site in substrate hydrolysis.     

Helicobacter pylori induces macrophage apoptosis by activation of arginase II

Helicobacter pylori infection induces innate immune responses in macrophages, contributing to mucosal inflammation and damage. Macrophage apoptosis is important in the pathogenesis of mucosal infections but has not been studied with H. pylori. NO derived from inducible NO synthase (iNOS) can activate macrophage apoptosis. Arginase competes with iNOS by converting L-arginine to L-ornithine. Since we reported that H. pylori induces iNOS in macrophages, we now determined whether this bacterium induces arginase and the effect of this activation on apoptosis. NF-kappa B-dependent induction of arginase II, but not arginase I, was observed in RAW 264.7 macrophages cocultured with H. pylori. The time course of apoptosis matched those of both arginase and iNOS activities. Surprisingly, apoptosis was blocked by the arginase inhibitors N(omega)-hydroxy-L-arginine or N(omega)-hydroxy-nor-L-arginine, but not by the iNOS inhibitor N-iminoethyl-L-lysine. These findings were confirmed in peritoneal macrophages from iNOS-deficient mice and were not dependent on bacterial-macrophage contact. Ornithine decarboxylase (ODC), which metabolizes L-ornithine to polyamines, was also induced in H. pylori-stimulated macrophages. Apoptosis was abolished by inhibition of ODC and was restored by the polyamines spermidine and spermine. We also demonstrate that arginase II expression is up-regulated in both murine and human H. pylori gastritis tissues, indicating the likely in vivo relevance of our findings. Therefore, we describe arginase- and ODC-dependent macrophage apoptosis, which implicates polyamines in the pathophysiology of H. pylori infection.     

In hepatocytes the regulation of NOS-2 activity at physiological l-arginine levels suggests a close link to the urea cycle

High-output synthesis of nitric oxide (NO) by the inducible isoform of NO-synthases (NOS-2) plays an important role in hepatic pathophysiological processes and may contribute to both organ protection and organ destruction during inflammatory reactions. As they compete for the same substrate, L-arginine, an interdependence of NOS-2 and arginase-1 has been repeatedly observed in cells where arginase-1 is cytokine-inducible. However, in hepatocytes, arginases are constitutively expressed and thus, their impact on hepatic NOS-2-derived NO synthesis as well as the influence of L-arginine influx via cationic amino acid transporters during inflammatory reactions are still under debate. Freshly isolated rat hepatocytes were cultured for 24h in the presence of various L-arginine concentrations with or without cytokine addition and nitrite and urea accumulation in culture supernatants was measured. We find that both, cytokine-induced NOS-2 and arginase activities strongly depend on extracellular L-arginine concentrations. When we competed for L-arginine influx via the cationic amino acid transporters by addition of L-lysine, we find a 60-70% inhibition of arginase activity without significant loss of NOS-2 activity. Addition of L-valine, as an arginase inhibitor, leads to a 25% increase in NO formation and an 80-90% decrease in arginase activity. Interestingly, product inhibition of arginase and competitive inhibition of CATs through the addition of L-ornithine leads to a highly significant increase in hepatocytic NOS-2 activity with a concomitant and complete abolishment of its dependence on extracellular L-arginine concentrations. In conclusion, hepatocytic NOS-2 activity shows a surprising pattern of dependence on exogenous L-arginine concentrations. Inhibition and competition experiments suggest a relatively tight link of NOS-2 and urea cycle activities. These data stress the hypothesis of a metabolon-like organization of the urea cycle together with NOS-2 in hepatocytes as excess L-ornithine will be metabolized to l-arginine and thereby increases NO production.     

Effect of L-ornithine on proliferative and cytotoxic T-cell responses in allogeneic and syngeneic mixed leukocyte cultures

The effect of L-ornithine on cytotoxic and proliferative responses in mixed leukocyte cultures has been analyzed. The activation of cytotoxic T lymphocytes (CTL) was strongly inhibited when 9 X 10(-3) M L-ornithine was added at the initiation of the cultures. The CTL precursor cells were not completely and irreversibly inactivated, however, since the cells generated normal cytotoxic activity if resuspended after 6 days in fresh culture medium together with a fresh set of stimulator cells. Experiments in microcultures with nylon-wool-nonadherent T-cell-enriched spleen cells as responder cells and "plastic adherent cells" as stimulator cells revealed that the cytotoxic responses were almost completely suppressed if ornithine was added within the first 20 hr but were only partially suppressed if ornithine was added after 48 hr. Also, ornithine had only a mild suppressive effect on proliferative responses in allogeneic and syngeneic mixed leukocyte cultures. The strong suppressive effect of the cytotoxic response was therefore not explained by a general toxic effect of L-ornithine on the responding cells in the culture. The addition of interleukin 2 (IL-2)-containing EL-4 supernatants did not prevent but rather enhanced the suppressive effect of L-ornithine. This indicated that the inhibitory effect was not (exclusively) expressed at the level of the IL-2-producing helper T cells. Since activated macrophages have been reported to secrete arginase, it appears that L-ornithine may be part of a regulatory circuit that selectively regulates the development of cytotoxic effector T cells.     

Manganese-dependent inhibition of human liver arginase by borate

Full activation of human liver arginase (EC 3.5.3.1), by incubation with 5 mM Mn2+ for 10 min at 60 degrees C, resulted in increased Vmax and a higher sensitivity of the enzyme to borate inhibition, with no change in the K(m) for arginine. Borate behaved as an S-hyperbolic I-hyperbolic non-competitive inhibitor and had no effect on the interaction of the enzyme with the competitive inhibitors L-ornithine (Ki = 2 +/- 0.5 mM), L-lysine (Ki = 2.5 +/- 0.4 mM), and guanidinium chloride (Ki = 100 +/- 10 mM). The pH dependence of the inhibition was consistent with tetrahedral B(OH)4- being the inhibitor, rather than trigonal B(OH)3. We suggest that arginase activity is associated with a tightly bound Mn2+ whose catalytic action may be stimulated by addition of a more loosely bound Mn2+, to generate a fully activated enzyme form. The Mn2+ dependence and partial character of borate inhibition are explained by assuming that borate binds in close proximity to the loosely bound Mn2+ and interferes with its stimulatory action. Although borate protects against inactivation of the enzyme by diethyl pyrocarbonate (DEPC), the DEPC-sensitive residue is not considered as a ligand for borate binding, since chemically modified species, which retain about 10% of enzymatic activity, were also sensitive to the inhibitor.     

Arginase activity is inhibited by L-NAME,both in vitro and in vivo

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.     

Arginase Activity is Inhibited by l -NAME,both In Vitro and In Vivo

The present study investigated the ability of the arginine analog L -NAME (N^ω-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.     

Allosteric inhibition of rat liver and kidney arginase by copper and mercury ions

Two isozyme forms of arginase are found in the rat. All arginases are metalloenzymes which require manganese for activity. Many arginases are activated by cobalt and nickel ions and inhibited by heavy metal ions. The purpose of this study was to compare the effect of other heavy metal ions on the rat liver isozyme (arginase I) and the rat kidney isozyme (arginase II). The activation and inhibition of arginase I and II by metal ions were different. However, both isozymes were strongly inhibited by cupric and mercuric ions. The inhibition of arginase I by cupric and mercuric ions was increased greatly by preincubation of the enzyme with the metal ions. However, preincubation of arginase II by cupric and mercuric ions had little effect on the inhibition of the enzyme. Under certain conditions the kinetics of the inhibition of both arginases I and II by cupric and mercuric ions was nonlinear allosteric.     

The involvement of tyrosine kinases, cyclic AMP/protein kinase A, and p38 mitogen-activated protein kinase in IL-13-mediated arginase I induction in macrophages: its implications in IL-13-inhibited nitric oxide production

In macrophages, L-arginine can be used by NO synthase and arginase to form NO and urea, respectively. Therefore, activation of arginase may be an effective mechanism for regulating NO production in macrophages through substrate competition. Here, we examined whether IL-13 up-regulates arginase and thus reduces NO production from LPS-activated macrophages. The signaling molecules involved in IL-13-induced arginase activation were also determined. Results showed that IL-13 increased arginase activity through de novo synthesis of the arginase I mRNA and protein. The activation of arginase was preceded by a transient increase in intracellular cAMP, tyrosine kinase phosphorylation, and p38 mitogen-activated protein kinase (MAPK) activation. Exogenous cAMP also increased arginase activity and enhanced the effect of IL-13 on arginase induction. The induction of arginase was abolished by a protein kinase A (PKA) inhibitor, KT5720, and was down-regulated by tyrosine kinase inhibitors and a p38 MAPK inhibitor, SB203580. However, inhibition of p38 MAPK had no effect on either the IL-13-increased intracellular cAMP or the exogenous cAMP-induced arginase activation, suggesting that p38 MAPK signaling is parallel to the cAMP/PKA pathway. Furthermore, the induction of arginase was insensitive to the protein kinase C and p44/p42 MAPK kinase inhibitors. Finally, IL-13 significantly inhibited NO production from LPS-activated macrophages, and this effect was reversed by an arginase inhibitor, L-norvaline. Together, these data demonstrate for the first time that IL-13 down-regulates NO production through arginase induction via cAMP/PKA, tyrosine kinase, and p38 MAPK signalings and underline the importance of arginase in the immunosuppressive activity of IL-13 in activated macrophages.     

Gene transfer with inducible nitric oxide synthase decreases production of urea by arginase in pulmonary arterial endothelial cells

Nitric oxide (NO) is a vasodilator produced from L-arginine (L-Arg) by NO synthase (NOS). Gene therapy for hypertensive disorders has been proposed using the inducible isoform of NOS (iNOS). L-Arg also can be metabolized to urea and L-ornithine (L-Orn) by arginase, and L-Orn can be metabolized to proline and/or polyamines, which are vital for cellular proliferation. To determine the effect of iNOS gene transfer on arginase, we transfected bovine pulmonary arterial endothelial cells (bPAEC) with an adenoviral vector containing the gene for iNOS (AdiNOS). As expected, NO production in AdiNOS bPAEC was substantially greater than in control bPAEC. Although urea production was significantly less in the AdiNOS bPAEC than in the control bPAEC, despite similar levels of arginase I protein, AdiNOS transfection of bPAEC had no effect on the uptake of L-Arg. Inhibiting NO production with Nomega-nitro-L-arginine methyl ester increased urea production, and inhibiting urea production with L-valine increased nitrite production, in AdiNOS bPAEC. The addition of L-Arg to the medium increased urea production by AdiNOS bPAEC in a concentration-dependent manner. Thus, in these iNOS-transfected bPAEC, the transfected iNOS and native arginase compete for a common intracellular pool of L-Arg. This competition for substrate resulted in impaired proliferation in the AdiNOS-transfected bPAEC. These findings suggest that the use of iNOS gene therapy for pulmonary hypertensive disorders may not only be beneficial through NO-mediated pulmonary vasodilation but also may decrease vascular remodeling by limiting L-Orn production by native arginase.     

Differential regulation of nitric oxide synthase-2 and arginase-1 by type 1/type 2 cytokines in vivo: granulomatous pathology is shaped by the pattern of L-arginine metabolism.

Type 2 cytokines regulate fibrotic liver pathology in mice infected with Schistosoma mansoni. Switching the immune response to a type 1-dominant reaction has proven highly effective at reducing the pathologic response. Activation of NOS-2 is critical, because type 1-deviated/NO synthase 2 (NOS-2)-deficient mice completely fail to control their response. Here, we demonstrate the differential regulation of NOS-2 and arginase type 1 (Arg-1) by type 1/type 2 cytokines in vivo and for the first time show a critical role for arginase in the pathogenesis of schistosomiasis. Using cytokine-deficient mice and two granuloma models, we show that induction of Arg-1 is type 2 cytokine dependent. Schistosome eggs induce Arg-1, while Mycobacterium avium-infected mice develop a dominant NOS-2 response. IFN-gamma suppresses Arg-1 activity, because type 1 polarized IL-4/IL-10-deficient, IL-4/IL-13-deficient, and egg/IL-12-sensitized animals fail to up-regulate Arg-1 following egg exposure. Notably, granuloma size decreases in these type-1-deviated/Arg-1-unresponsive mice, suggesting an important regulatory role for Arg-1 in schistosome egg-induced pathology. To test this hypothesis, we administered difluoromethylornithine to block ornithine-aminodecarboxylase, which uses the product of arginine metabolism, L-ornithine, to generate polyamines. Strikingly, granuloma size and hepatic fibrosis increased in the ornithine-aminodecarboxylase-inhibited mice. Furthermore, we show that type 2 cytokine-stimulated macrophages produce proline under strict arginase control. Together, these data reveal an important regulatory role for the arginase biosynthetic pathway in the regulation of inflammation and demonstrate that differential activation of Arg-1/NOS-2 is a critical determinant in the pathogenesis of granuloma formation.     

Arginase II inhibited lipopolysaccharide-induced cell death by regulation of iNOS and Bcl-2 family proteins in macrophages

Arginase II catalyzes the conversion of arginine to urea and ornithine in many extrahepatic tissues. We investigated the protective role of arginase II on lipopolysaccharide-mediated apoptosis in the macrophage cells. Adenoviral gene transfer of full length of arginase II was performed in the murine macrophage cell line RAW264.7. The role of arginase II was investigated with cell viability, cytoplasmic histone-associated DNA fragmentation assay, arginase activity, nitric oxide production, and Western blot analysis. Arginase II is localized in mitochondria of macrophage cells, and the expression of arginase II was increased by lipopolysaccharide (LPS). LPS significantly increased cell death which was inhibited by AMT, a specific inducible nitric oxide synthase (iNOS) inhibitor. In contrast, LPS-induced cell death and nitric oxide production were increased by 2-boronoethyl-L-cysteine, a specific inhibitor of arginase. Adenoviral overexpression of arginase II significantly inhibited LPS-induced cell death and cytoplasmic histone-associated DNA fragmentation. LPS-induced iNOS expression and poly ADP-ribose polymerase cleavage were significantly suppressed by arginase II overexpression. Furthermore, arginase II overexpression resulted in a decrease in the Bax protein level and the reverse induction of Bcl-2 protein. Our data demonstrated that inhibition of NO production by arginase II may be due to arginine depletion as well as iNOS suppression though its reaction products. Moreover, arginase II plays a protective role of LPS-induced apoptosis in RAW264.7 cells.     

Characterization of ornithine decarboxylase of tobacco cells and tomato ovaries.

Some characteristics of L-ornithine decarboxylase of tomato ovaries and tobacco cells are described. The enzyme has a pH optimum of 8.0. It requires pyridoxal phosphate and thiol reagent (dithiothreitol) for activity. It is specific for L-ornithine and has an apparent Km of 1.4 X 10-4 M. It has an apparent molecular weight of 107000. Putrescine inhibited the activity in vitro. Spermidine and spermine also inhibit the enzyme, but less effectively. It is concluded that the enzyme is similar to that of mammalian origin and likewise fulfils a function related to cell proliferation.     

Arginine deprivation and tumour cell death: Arginase and its inhibition

Arginase treatment of cell cultures reduced arginine in the medium to ~ micromolar levels within 5–30 min, and proved as effective as arginine-free medium (AFM) prepared by formulation. The enzyme was heat stable and as active at pH 7.2 as at pH 9.9. It persisted in culture for at least 3 days with only a small diminution in its speed of action, and still actively destroyed arginine after 6 days, since arginine supplementation failed to rescue viable cells.Addition of L-norvaline, an inhibitor of arginase, rescued cells from arginase-induced deprivation. Its efficacy at low concentrations was short-lived (probably < 1 day), while at higher concentrations it did not appear to inhibit completely the enzyme. However, L-norvaline at these same levels also slowed the growth of positive non-enzyme treated controls receiving the normal arginine levels. Thus the difference in this growth indicated that arginase was more inhibitory than cursory examination of initial kinetic data suggested. It also agreed with the inhibition of arginase in the ornithine assay used to measure biochemically enzyme activity. We conclude that norvaline partially but not completely antagonises arginase activity, which allows cell rescue in a dose-dependent manner between 0.4 and 4 mM, but cannot be used above about 2 mM without exhibiting a general non-specific interference of cell growth of its own, although no evidence of cell toxicity was observed in either AFM or arginine-containing medium. L-ornithine, the product of arginase that inhibits the enzyme by a feedback mechanism, had no inhibitory effect on arginase over a similar concentration range.     

Expression, purification and characterization of arginase from Helicobacter pylori in its apo form

Arginase, a manganese-dependent enzyme that widely distributed in almost all creatures, is a urea cycle enzyme that catalyzes the hydrolysis of L-arginine to generate L-ornithine and urea. Compared with the well-studied arginases from animals and yeast, only a few eubacterial arginases have been characterized, such as those from H. pylori and B. anthracis. However, these enzymes used for arginase activity assay were all expressed with LB medium, as low concentration of Mn(2+) was detectable in the medium, protein obtained were partially Mn(2+) bonded, which may affect the results of arginase activity assay. In the present study, H. pylori arginase (RocF) was expressed in a Mn(2+) and Co(2+) free minimal medium, the resulting protein was purified through affinity and gel filtration chromatography and the apo-form of RocF was confirmed by flame photometry analysis. Gel filtration indicates that the enzyme exists as monomer in solution, which was unique as compared with homologous enzymes. Arginase activity assay revealed that apo-RocF had an acidic pH optimum of 6.4 and exhibited metal preference of Co(2+)>Ni(2+)>Mn(2+). We also confirmed that heat-activation and reducing regents have significant impact on arginase activity of RocF, and inhibits S-(2-boronoethyl)-L-Cysteine (BEC) and Nω-hydroxy-nor-Arginine (nor-NOHA) inhibit the activity of RocF in a dose-dependent manner.     

Yeast epiarginase regulation,an enzyme-enzyme activity control: identification of residues of ornithine carbamoyltransferase and arginase responsible for enzyme catalytic and regulatory activities

In the presence of ornithine and arginine, ornithine carbamoyltransferase (OTCase) and arginase form a one-to-one enzyme complex in which the activity of OTCase is inhibited whereas arginase remains catalytically active. The mechanism by which these nonallosteric enzymes form a stable complex triggered by the binding of their respective substrates raises the question of how such a cooperative association is induced. Analyses of mutations in both enzymes identify residues that are required for their association, some of them being important for catalysis. In arginase, two cysteines at the C terminus of the protein are crucial for its epiarginase function but not for its catalytic activity and trimeric structure. In OTCase, mutations of putative ornithine binding residues, Asp-182, Asn-184, Asn-185, Cys-289, and Glu-256 greatly reduced the affinity for ornithine and impaired the interaction with arginase. The four lysine residues located in the SMG loop, Lys-260, Lys-263, Lys-265, and Lys-268, also play an important role in mediating the sensitivity of OTCase to ornithine and to arginase and appear to be involved in transducing and enhancing the signal given by ornithine for the closure of the catalytic domain.     

Mechanistic and metabolic inferences from the binding of substrate analogues and products to arginase

Arginase is a binuclear Mn(2+) metalloenzyme that catalyzes the hydrolysis of L-arginine to L-ornithine and urea. X-ray crystal structures of arginase complexed to substrate analogues N(omega)-hydroxy-L-arginine and N(omega)-hydroxy-nor-L-arginine, as well as the products L-ornithine and urea, complete a set of structural "snapshots" along the reaction coordinate of arginase catalysis when interpreted along with the X-ray crystal structure of the arginase-transition-state analogue complex described in Kim et al. [Kim, N. N., Cox, J. D., Baggio, R. F., Emig, F. A., Mistry, S., Harper, S. L., Speicher, D. W., Morris, Jr., S. M., Ash, D. E., Traish, A. M., and Christianson, D. W. (2001) Biochemistry 40, 2678-2688]. Taken together, these structures render important insight on the structural determinants of tight binding inhibitors. Furthermore, we demonstrate for the first time the structural mechanistic link between arginase and NO synthase through their respective complexes with N(omega)-hydroxy-L-arginine. That N(omega)-hydroxy-L-arginine is a catalytic intermediate for NO synthase and an inhibitor of arginase reflects the reciprocal metabolic relationship between these two critical enzymes of L-arginine catabolism.     

Arginase activity and other cellular events associated with epidermal hyperplasia

The unknown biochemical role of arginase in epidermal metabolism was probed by examining the association of elevated arginase activty with epidermal hyperplasia and hyperkeratinization. Epidermal hyperplasia as induced experimentally by topical application of 1-decanol to the right side of male hairless mice while the contralateral side served as control. Arginase activity, incorporation of ³ H-thymidine into DNA, DNA and protein content were measured in the separated control and experimental epidermis six hours and on days 1 through 5 and 7 after 1-decanol application. After six hours, the epidermis appears damaged histologically, and DNA synthesis is inhibited. By day 1, incoporation of ³ H-thymidine into DNA is elevated and a new hyperplastic epidermis has formed beneath the original epidermis. Epidermal arginase is elevated two through seven days after 1-decanol application and always is associated with continuing epidermal hyperplasia. The stimulation of DNA synthesis, which parallels the induction of epidermal hyperplasia by 1-decanol, precedes the induction of epidermal arginase activity. An attempt to relate these results with polyamine synthesis and other metabolic events is made.