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Substrate kinetics of arginase were investigated in normal and sciatectomized gastrocnemius muscle of frog. Sciatectomized muscle arginase showed 47% elevation in V while Km showed no change. Sciatectomic activation of muscle arginase accounted for the stepped up urea levels which may be of significant use in combating ammonia toxicity and maintaining chemohomeostasis.
The molecular weight of arginase, determined by gel filtration of frog (Rana tigerina) liver and kidney extracts, varied with the protein concentration. It increased from about 25-27 KDa in dilute solutions to about 92-96 KDA in concentrated extracts. In addition, an arginase with a molecular weight of 45-49 KDA appeared at intermediate concentrations. The results suggest the probable aggregation of arginase monomers to yield the dimeric and tetrameric forms of the enzyme at high protein concentration in extracts.
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.
Hepatic L-arginase from the Mus booduga (Gray) was purified and its kinetic characteristics were investigated. The enzyme was not adsorbed on DEAE-cellulose, but was retained on CM-cellulose column at pH 7.2. The Michaelis-Menten constant was 8.3 mM for L-arginine and was independent of pH in the range of 7.5-10.5. L-arginine concentrations as high as 0.4 M did not exert substrate inhibition in the pH range 7.4-10.0. Manganese was required at a concentration of 0.05 M for full activation of the enzyme. L-ornithine and L-lysine inhibited the enzyme competitively with inhibitory constants of 1.9 mM and 3.7 mM respectively. Several properties of the L-arginase from Mus booduga clearly identify it as an enzyme similar to ureotelic basic arginases from mammalian liver.
Regulation of macrophage activities in response to inflammatory stimuli must be finely tuned to promote an effective immune response while, at the same time, preventing damage to the host. Our lab and others have previously shown that macrophage-stimulating protein (MSP), through activation of its receptor RON, negatively regulates NO production in response to IFN-gamma and LPS by inhibiting the expression of inducible NO synthase (iNOS). Furthermore, activated macrophages from mice harboring targeted mutations in RON produce increased levels of NO both in vitro and in vivo, rendering them more susceptible to LPS-induced endotoxic shock. In this study, we demonstrate that stimulation of murine peritoneal macrophages with MSP results in the RON-dependent up-regulation of arginase, an enzyme associated with alternative activation that competes with iNOS for the substrate L-arginine, the products of which are involved in cell proliferation and matrix synthesis. Expression of other genes associated with alternative activation, including scavenger receptor A and IL-1R antagonist, is also up-regulated in MSP-stimulated murine macrophages. Stimulation of cells with IFN-gamma and LPS blocks the ability of MSP to induce arginase activity. However, pretreatment of cells with MSP results in the up-regulation of arginase and inhibits their ability to produce NO in response to IFN-gamma and LPS, even in the presence of excess substrate, suggesting that the inhibition of NO by MSP occurs primarily through its ability to regulate iNOS expression.
Helicobacter pylori infects approximately half the human population. The outcomes of the infection range from gastritis to gastric cancer and appear to be associated with the immunity to H. pylori. Patients developing nonatrophic gastritis present a Th1 response without developing protective immunity, suggesting that this bacterium may have mechanisms to evade the immune response of the host. Several H. pylori proteins can impair macrophage and T cell function in vitro through mechanisms that are poorly understood. We tested the effect of H. pylori extracts and live H. pylori on Jurkat cells and freshly isolated human normal T lymphocytes to identify possible mechanisms by which the bacteria might impair T cell function. Jurkat cells or activated T lymphocytes cultured with an H. pylori sonicate had a reduced proliferation that was not caused by T cell apoptosis or impairment in the early T cell signaling events. Instead, both the H. pylori sonicate and live H. pylori induced a decreased expression of the CD3zeta-chain of the TCR. Coculture of live H. pylori with T cells demonstrated that the wild-type strain, but not the arginase mutant rocF(-), depleted L-arginine and caused a decrease in CD3zeta expression. Furthermore, arginase inhibitors reversed these events. These results suggest that H. pylori arginase is not only important for urea production, but may also impair T cell function during infection.
BACKGROUND: Arginase is an enzyme of the urea cycle, and one of the two isoenzymes is the liver-type enzyme. We examined serum autoantibodies to this liver-type enzyme in patients with hepatitis. METHODS: Antibodies to recombinant human liver-type arginase were measured by ELISA in 95 patients and 55 healthy controls. RESULTS: The mean absorbance values in the ELISA assays of patients with definite autoimmune hepatitis (n = 11; P <0.0001), probable autoimmune hepatitis (n = 31; P <0.0001), and hepatitis C (HCV; n = 20; P <0.01) were significantly different from those of healthy controls, but the values in patients with hepatitis B (HBV; n = 23) and other autoimmune diseases (n = 10) were not significantly different from those of healthy controls. When the cutoff was fixed at the upper 95th percentile of the absorbance value in healthy controls, positive reactions were found in 18.2%, 32.3%, 20.0%, 13. 0%, and 10.0% of patients with definite autoimmune hepatitis, probable autoimmune hepatitis, HCV hepatitis, HBV hepatitis, and other autoimmune diseases, respectively. All of these positive reactions were abolished by inhibition of serum with recombinant antigen. The specificity and sensitivity of this ELISA were 96% and 29%, respectively. The intraassay and interassay coefficients of variation were 2.3-7.5% and 9.8-11%, respectively. There was no relationship between these antibodies and anti-nuclear, anti-smooth muscle, or anti-cytochrome P450IID6 antibodies. CONCLUSIONS: The ELISA for anti-liver-type arginase autoantibody improved the detectability of autoimmune hepatitis when compared with established assays for liver-specific autoantibodies.
The effect of L-arginine on the arginase activity and polyamine levels was studied in the pancreas of normal and diabetic rats (120 mg/kg alloxan, i.v.). Four groups were formed (10 male adults per group). I-Control-0.154 M NaCl. II-Diabetic-0.154 M NaCl (96 h after alloxan). III-Control plus 10 mM L-arginine IV-Diabetics plus 10 mM L-arginine. Rats were sacrificed 20 min after L-arginine injection. Glucose in serum and dry weight, proteins, arginase activity and polyamines (HPLC) in pancreas were measured. Higher ratio mg protein/mg dry weight and arginase induction was observed for groups III and IV. Putrescine was low as a consequence of diabetes but restored with L-arginine. The concentrations of spermidine and spermine were lower. These results may suggest that arginine is metabolized to putrescine in the pancreas and that polyamines may be utilized in regenerating processes or for recovering the endocrine pancreatic function.
It has been studied an action of chronic urea introduction (40 mg/kg, 14 and 28 days) as arginase inhibitor on nonoxidative (arginase activity, urea, polyamines content) and oxidative (NOS activity, nitrite- and nitrate-anion content) metabolism of L-arginine in aorta, heart, plasma and erythrocytes of SHR. It has been shown that urea is an inhibitor of not arginase only, but also ornitinedecarboxilase (ODK) reactions, limiting L-arginine consumption for urea and polyamines synthesis and thus facilitating its utilization for nitric oxide synthesis by NOS. These exogenous effects of urea are not accompanied by amelioration of tissue ischaemization within cardiovascular system. It has been shown that exogenous urea down regulate blood pressure without any normalization of endothelim-dependent reactions of smooth muscle cells on acetylcholine in SHR.
Previous studies have shown that peripheral vestibular damage causes long-term neurochemical changes in the hippocampus which may be related to spatial memory deficits. Since recent studies have also demonstrated deficits in non-spatial object recognition memory following vestibular lesions, the aim of the present study was to extend these investigations into the perirhinal cortex (PRC), which is known to be important for object recognition, and the related entorhinal cortex (EC). We examined the effects of unilateral vestibular deafferentation (UVD) on the expression of four enzymes associated with neuronal plasticity, neuronal nitric oxide synthase (nNOS), endothelial nitric oxide synthase (eNOS), arginase I and arginase II (AI and II), in the rat EC and PRC using Western blotting. Tissue was collected at 10 hs, 50 hs and 2 weeks post-UVD. In the EC and PRC, nNOS protein expression decreased on the contralateral side at 2 weeks post-UVD but not before. At the same time, eNOS protein expression increased in both regions on the contralateral side. In the EC, AII protein expression increased on the ipsilateral side at 2 weeks post-UVD. In the PRC, AI increased and decreased on the contralateral and ipsilateral sides (respectively) at 2 weeks post-UVD. AII showed a bilateral increase in the PRC at 2 weeks post-UVD. These results demonstrate changes in NOS and arginase protein expression in the PRC and EC following UVD, which are unlikely to be due to the initial severity of the vestibular syndrome because they develop well after vestibular compensation has taken place. Neurochemical changes in these regions of the medial temporal lobe may be implicated in the development of object recognition deficits that contribute to cognitive dysfunction following peripheral vestibular damage.
Arginase from Saccharomyces cerevisiae has long been known to be a metal ion-requiring enzyme as it requires heating at 45 degrees C in the presence of 10 mM Mn2+ for catalytic activation. Metals are also thought to play a structural role in the enzyme, but the identity of the structural metal and its precise structural role have not been defined. Analysis of the metal ions that bind to yeast arginase by atomic absorption spectroscopy reveals that there is a weakly associated Mn2+ that binds to the trimeric enzyme with a stoichiometry of 1.04 +/- 0.05 mol of Mn2+ bound per subunit and an apparent K'D value of 26 microM at pH 7.0 and 4 degrees C. A more tightly associated Zn2+ ion can only be removed by dialysis against chelating agents. In occasional preparations, this site contained some Mn2+; however, Zn2+ and Mn2+ together bind to high affinity sites with a stoichiometry of 1.14 +/- 0.25/mol of subunit. Both the loosely associated catalytic Mn2+ ion and the more tightly associated structural Zn2+ ion confer stability to the enzyme. Removal of the weakly bound Mn2+ ion results in a 3 degree C decrease in the midpoint of the thermal transition (T 1/2) (from 57 by 54 degrees C) as monitored by UV difference absorption spectroscopy. Removal of the tightly bound Zn2+ ion produces a 19 degrees C decrease in T 1/2 (to 38 degrees C). Similar results are obtained by circular dichroism measurements. When the Zn2+ ion is removed, the steady-state fluorescence intensity increases 100% as compared to the holoenzyme, with a shift in the emission maximum from 337 to 352 nm. This suggests that in the folded trimeric metalloenzyme, the tryptophan fluorescence is quenched and that upon removal of the structural metal, the quenching is relieved as tryptophan residues become exposed to more polar environments. Equilibrium sedimentation experiments performed after dialysis of the enzyme against EDTA demonstrate that arginase exists in a reversible monomer-trimer equilibrium, in the absence of metal ions, with a KD value of 5.05 x 10(-11) M2. In contrast, the native enzyme exists as a trimer with no evidence of dissociation when Mn2+ and Zn2+ are present (Eisenstein, E., Duong, L.T., Ornberg, R. L., Osborne, J.C., Jr., and Hensley, P. (1986) J. Biol. Chem. 261, 12814-12819). In summary, the study presented here demonstrates that binding of a weakly bound Mn2+ ion confers catalytic activity. In contrast, binding of a more tightly associated Zn2+ ion confers substantial stability to the tertiary and quaternary structure of the enzyme.
Genes for urea cycle enzymes including liver-type arginase are expressed mainly in the liver and are regulated developmentally, nutritionally, and hormonally in a coordinated manner. The promoter region of the rat arginase gene was investigated with an in vitro transcription system using nuclear extracts prepared from rat tissues. Accurate initiation of the transcription in liver nuclear extracts was confirmed by runoff analysis and S1 nuclease mapping. The arginase promoter was transcribed more efficiently in liver nuclear extracts than in brain extracts, reproducing the in vivo tissue specificity qualitatively. Analysis of deletion mutants of the 5'-flanking region in liver nuclear extracts revealed a positive regulatory region spanning nucleotides -90 to -51 relative to the transcription start site. Overlapping this region, two protected areas were detected by DNase I footprinting. Competition analysis with synthetic oligonucleotides showed that the more downstream protected area was occupied, in a mutually exclusive manner, by two factors each related to CTF/NF-1 and Sp1. The other more upstream protected area was recognized by a factor related to the liver-enriched transcription factor C/EBP, which was recently shown to interact with regulatory regions of two other urea cycle enzyme genes.
Bull testis arginase was about 1,000 times purified. The molecular weight was estimated to 120,000 (+/- 250). The enzyme was an anionic protein. Km was determined to be 3.4 mM. According to electrophoretic mobility and affinity to DEAE-cellulose as well as to the immunological properties, the arginase isolated from bull testis may be identified as arginase A1.
Liver-type arginase (EC 3.5.3.1) catalyzes the last step of urea synthesis and is expressed specifically in the liver of ureotelic animals. Expression of liver arginase is developmentally and hormonally regulated in coordination with other urea cycle enzymes. The gene for the rat enzyme was cloned and the structure determined. This gene is 12 kilobases long and is split into eight exons. All of the splice donor and acceptor sites conform to the GT/AG rule. The transcription initiation site was determined by nuclease S1 mapping and primer extension. A "TATA box"-like sequence and a "CAAT box"-like sequence are present 27 and 60 bases upstream from the cap site, respectively. Upstream and downstream from the cap site, there are several sets of direct repeats and inverted repeats and several sequences resembling the transcription factor Sp1 binding sites, the enhancer core sequences, the glucocorticoid receptor binding sites, the 12-O-tetradecanoylphorbol-13-acetate responsive elements, and the putative elements for liver-specific expression of albumin genes. A 15-nucleotide sequence in the 5'-flanking region of the arginase gene is highly homologous with sequences in the 5'-flanking regions of the genes for rat ornithine carbamoyltransferase (EC 2.1.3.3) and for human argininosuccinate synthetase (EC 6.3.4.5), other two enzymes of the urea cycle.
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.
Arginase (EC 3.5.3.1) catalyzes the last step of urea synthesis in the liver of ureotelic animals. The nucleotide sequence of rat liver arginase cDNA, which was isolated previously (Kawamoto, S., Amaya, Y., Oda, T., Kuzumi, T., Saheki, T., Kimura, S., and Mori, M. (1986) Biochem. Biophys. Res. Commun. 136, 955-961) was determined. An open reading frame was identified and was found to encode a polypeptide of 323 amino acid residues with a predicted molecular weight of 34,925. The cDNA included 26 base pairs of 5'-untranslated sequence and 403 base pairs of 3'-untranslated sequence, including 12 base pairs of poly(A) tract. The NH2-terminal amino acid sequence, and the sequences of two internal peptide fragments, determined by amino acid sequencing, were identical to the sequences predicted from the cDNA. Comparison of the deduced amino acid sequence of the rat liver arginase with that of the yeast enzyme revealed a 40% homology.
Incubation of murine spleen and bone marrow cells with homogeneously purified interleukin 3 (IL-3) results in increased urea and ornithine production. This phenomenon is explained by a marked enhancement of arginase activity, assessed by transformation of labeled arginine into labeled urea during culture and by intracellular arginase assay. The enhancement of enzymatic activity is evident after 12 hours of incubation with IL-3, reaching a maximum after 24 hours. It is (1) dose dependent, (2) restricted to cells from hematopoietic organs (i.e., spleen and bone marrow), and (3) independent of cell proliferation, since irradiation of bone marrow cells does not abolish the arginase enhancing effect of IL-3 in spite of complete inhibition of proliferation. Furthermore, this new activity is specific to IL-3, since other colony-stimulating factors, i.e., granulocyte-macrophage colony-stimulating factor (GM-CSF) and L cell-derived colony-stimulating factor (L-CSF), do not induce it.
Arginase activity was studied in the brain and other tissues of the rat at the different periods of neurinoma growth. The activity of the enzyme was considerably activated in the affected hemisphere on the 4th day, in the skin of the head and thigh on the 6th day after tumor transplantation. Elevation in arginase activity in the neoplasm itself was recorded on the 16th day. The data obtained point to the physiological significance of arginase during neurinoma growth.