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PubMed1996

Delivery of cytosolic liver arginase into the mitochondrial matrix space: a possible novel site for gene replacement therapy.

As a toxic metabolic byproduct in mammals, excess ammonia is converted into urea by a series of five enzymatic reactions in the liver that constitute the urea cycle. A portion of this cycle takes place in the mitochondria, while the remainder is cytosolic. Liver arginase (L-arginine ureahydrolase, A1) is the fifth enzyme of the cycle, catalyzing the hydrolysis of arginine to ornithine and urea within the cytosol. Patients deficient in this enzyme exhibit hyperargininemia with episodic hyperammonemia and long-term effects of mental retardation and spasticity. However, the hyperammonemic effects are not so catastrophic in arginase deficiency as compared to other urea cycle defects. Earlier studies have suggested that this is due to the mitigating effect of a second isozyme of arginase (AII) expressed predominantly in the kidney and localized within the mitochondria. In order to explore the curious dual evolution of these two isozymes, and the ways in which the intriguing, aspects of AII physiology might be exploited for gene replacement therapy of AI deficiency, the cloned cDNA for human AI was inserted into an expression vector downstream from the mitochondrial targeting leader sequence for the mitochondrial enzyme ornithine transcarbamylase and transfected into a variety of recipient cell types. AI expression in the target cells was confirmed by northern blot analysis, and competition and immunoprecipitation studies showed successful translocation of the exogenous AI enzyme into the transfected cell mitochondria. Stability studies demonstrated that the translocated enzyme had a longer half-life than either native cytosolic AI or mitochondrial AII. Incubation of the transfected cells with increasing amounts of arginine produced enhanced levels of mitochondrial AI activity, a substrate-induced effect that we have previously seen with native AII but never AI. Along with exploring the basic biological questions of regulation and subcellular localization in this unique dual-enzyme system, these results suggest that the mitochondrial matrix space may be a preferred site for delivery of enzymes in gene replacement therapy.

Arginase↗

PubMed1984

Precocious induction of arginase in primary cultures of fetal rat hepatocytes.

Fetal rat hepatocytes were isolated and cultured in primary culture to investigate activity changes of arginase under defined conditions. In hormone-free medium, cultured cells maintained the enzyme activity at levels equal to that of freshly isolated cells for at least 4 d. Arginase activity could be induced by dexamethasone in hepatocytes isolated from 16.5-d-old fetuses although cells were competent to respond to glucagon only at the stage of 18.5 d. The combination of the two hormones induced greater levels of arginase activity than the individual compounds. These findings indicate that glucocorticoid and glucagon receptors appear early and sequentially before birth and reveal that cultured fetal hepatocytes provide a suitable system for the investigation of the role of hormones in the initiation of enzyme synthesis.

Animals↗

PubMed1978

Subunit interactions and immobilised dimers of human liver arginase.

Incubation of soluble human liver arginase (L-arginine amidinohydrolase, EC 3.5.3.1) with p-hydroxymercuribenzoate resulted in the dissociation of the enzyme into active dimers. Addition of 2-mercaptoethanol resulted in the regeneration of the tetrameric enzyme. When arginase, bound covalently to nylon, was incubated with p-hydroxymercuribenzoate, matrix-bound dimers were obtained. Incubation of these species with 2-mercaptoethanol resulted in stable, unmodified dimers. Based on this dissociation of arginase, a model with D2-symmetry is suggested for this enzyme. The specific activity, the Km value for arginine, pH optimum and the inhibition constants for ornithine and lysine were determined for monomeric, dimeric and tetrameric forms. It is concluded that the behaviour of the active sites of the monomers is not substantially altered by the interaction of these species in the oligomeric molecule.

Arginase↗

PubMed1975

Early diagnosis of myocardial infarction by arginase activity determination.

In blood serum of healthy persons the activity of arginase (EC 3.5.3.1) is very low, whereas in patients with myocardial infarction it increases within a few hours after the first attack of coronary pain, and returns to normal values after 3-5 days. No increase of arginase activity was observed in sera of patients with angina pectoris, coronary insufficiency or cardiac failure. Determination of arginase activity in blood serum may serve as a useful test in early differential diagnosis of myocardial infarction.

Angina Pectoris↗

PubMed1976

Ultramicromethod for the determination of human arginase in the presence of urea.

A technique for arginase determination in body fluids in the presence of urea is described. [14C]Arginine is hydrolysed by arginase to [14C]urea and ornithine. [14C]Urea is separated with paper chromatography and measured in a liquid scintillation counter. The experimental conditions including the pH, substrate concentration, activator, solvent for chromatography, urea inhibition, and arginase in hemolysates, are discussed.

Arginase↗

PubMed1995

Inhibition of arginase by NG-hydroxy-L-arginine in alveolar macrophages: implications for the utilization of L-arginine for nitric oxide synthesis.

The hypothesis was investigated that the nitric oxide (NO) synthase intermediate, NG-hydroxy-L-arginine (HOArg), is an arginase inhibitor in rabbit or rat alveolar macrophages. Exogenously applied HOArg strongly inhibited the arginase activity present in these cells (IC50 > or = 15 microM), and attenuated L-[3H]arginine transport (IC50 > or = 500 microM) in rabbit alveolar macrophages. Moreover, up to 37 microM HOArg were detected in the conditioned medium, but not in the lysate, of rat alveolar macrophages exposed to bacterial lipopolysaccharide for 18 h. HOArg may thus be a potent endogenous arginase inhibitor in these cells which increases the availability of L-arginine for NO biosynthesis.

Amino Acid Oxidoreductases↗

PubMed1996

The cloning, expression and crystallisation of a thermostable arginase.

The gene for the thermostable arginase from the thermophilic bacterium 'Bacillus caldovelox' has been cloned and sequenced. Expression of recombinant arginase at high levels has been achieved in E. coli using an inducible T7 RNA polymerase-based system. A facile purification procedure incorporating a heat-treatment step yielded 0.2 g of recombinant arginase per litre of induced culture. The kinetic properties of the purified recombinant protein are essentially identical to the native enzyme. The recombinant protein has been crystallised and one crystal form is isomorphous to crystals of the native protein.

Amino Acid Sequence↗

PubMed1991

Resolution of multiple forms of bovine liver arginase by chromatofocusing.

1. Bovine liver arginase could be resolved into three distinct peaks by chromatofocusing in the pH range 7-4. 2. In other experimental systems the enzyme appeared to consist of a single active component. 3. Sodium dodecylsulphate-polyacrylamide gel electrophoresis revealed a single band which could be assigned to arginase, with no indication of inherent or protease-induced multiplicity. 4. Lineweaver-Burk plots for arginine were linear over a wide concentration range, as were Dixon plots for reversible inhibitors. 5. Covalent inhibition by 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide gave semilogarithmic plots of residual activity vs time which were strictly linear. 6. It was concluded that the enzyme was homogeneous with respect to subunit size and kinetic behaviour, but heterogeneous with respect to molecular charge. 7. The charge heterogeneity may have kinetic and regulatory implications, as previously suggested for mouse liver arginase [Z. Spolarics and J. S. Bond (1988) Archs Biochem. Biophys. 260, 469-479].

Animals↗

PubMed1980

Arginase production by peritoneal macrophages: a new assay.

The arginase activity of murine mononuclear cells was assessed with a new highly sensitive radioassay. Adherent mononuclear cells obtained by peritoneal lavage from normal mice contain significant quantities of enzyme. This was released in culture and continued synthesis could be blocked with cycloheximide. Arginase activity was not found in adherent or non-adherent mononuclear cells from the spleen. Peritoneal cells obtained from mice infected with P. berghei or from mice injected with LPS intraperitoneally had higher arginase activity than cells from control mice.

Animals↗

PubMed1993

Study of activities of arginase, hexosaminidase, and leucine aminopeptidase in prostate fluid.

Our previous studies demonstrated that extracts of the cancerous prostate tissues exhibit increased activities of arginase and hexosaminidase and decreased activity of leucine aminopeptidase. In an endeavor to devise a reliable, noninvasive and cost effective method for the diagnosis of prostate carcinoma, prostate fluids collected from 123 patients were analyzed for arginase, hexosaminidase, and leucine aminopeptidase. Based on the clinical diagnoses, the patients were divided into four groups: 13 men with chronic prostatitis, 29 with no recognized prostatic lesions, 63 with benign prostatic hyperplasia, and 18 with prostate cancer. The prostate fluids had no detectable arginase activity. The means of the total (units/mL) and the specific activities (units/mg of protein) of hexosaminidase and leucine aminopeptidase varied, albeit there were no significant differences in the four groups of patients recruited in this study. It was concluded that the profiles of enzymes in prostate fluids might differ from that in the secreting prostate tissues and that the results of enzyme analysis in prostate fluids for the diagnosis of cancer should be interpreted with caution.

Adolescent↗

PubMed1982

Evidence for cooperative effects in human liver arginase.

The reaction kinetics of human liver arginase (L-arginine amidinohydrolase, EC 3.5.3.1) in terms of arginine concentration is strikingly altered by varying the pH. Lowering the pH from the optimum (9.5) toward a more physiological value (7.5) there is a transition from hyperbolic to sigmoidal kinetics. The cooperative effects are observed in the presence and absence of the product ornithine. Dimers of arginase exhibit typical Michaelis-Menten kinetics even in the presence of ornithine. Dimer-dimer interactions are suggested to explain the kinetic properties of arginase at pH 7.5.

Arginase↗

PubMed1982

Kinetic properties of arginase from Xenopus laevis.

1. Arginase from the liver of Xenopus laevis has a Michaelis constant (Km) for arginine of 42 mM, a minor component having a Km of 29 mM. The Km is independent of pH in the range of 7.0-11.0 and also independent of manganese ion concentration. 2. Manganese is required for activation, showing an optimum at 50 mM with inhibition at higher concentrations. 3. The enzyme is inhibited by high concentrations of the substrate L-arginine and by L-lysine and L-ornithine in a competitive manner with respect to arginine. 4. In its Michaelis constant for arginine and inhibition by high substrate concentration, Xenopus liver arginase resembles "ureotelic" arginases.

Animals↗

PubMed1996

Hormonal and nutritional modulation of hepatic arginase activity in growing cattle.

The hormonal and nutritional modulation of hepatic arginase activity (HARG) was characterized in growing cattle in two studies. In the first study, 20 steers (initial weight, 182 +/- 2 kg) were assigned in equal numbers to either Synovex-S (SYN) (ear implant), recombinant bovine somatotropin (Somavubove; SbV; 0.1 mg/kg intramuscularly daily), SYN + SbV, or nothing (control). Steers were individually fed, for 56 d, a concentrate (80% diet dry matter [DM]) and silage (20% diet DM) diet providing 20 g of crude protein (CP) and 252 kcal metabolizable energy (ME) per kg body weight0.75. On Day 57, samples of liver were obtained at slaughter and subsequently assayed for HARG by the incubation of a tissue homogenate for 2 hr with 250 mM arginine, with and without Mn2+ and heat activation, and the measurement of the resulting urea. HARG was uniformly increased by divalent cation (Mn2+) and heating. SYN had no effect on HARG, whereas SbV treatment resulted in an overall 34% decrease in HARG. Plasma urea nitrogen (PUN) was decreased by SbV but not consistently affected by SYN. In the second study, 16 steers (avg. initial weight, 284 +/- 5 kg) were initially fed a concentrate basal diet consisting of 11% CP and 1.96 Mcal ME for 21 d. Steers were then assigned to one of four dietary treatments (6.4 kg DM/hr per day) in a factorial arrangement of high and low CP (8 and 14%) and two levels of energy (1.96 and 2.67 Mcal ME/kg of diet DM) for 210 d and slaughtered. HARG and PUN were higher in steers fed 14% CP but were lower at each level of CP fed at the higher level of ME. The data suggest that hormonal repartitioning compounds and diet composition may modulate nitrogen metabolism by affecting the activity of arginase in the liver as well as by affecting the total content of arginase in association with changes in organ size.

Animal Nutritional Physiological Phenomena↗

PubMed1994

An enzymatic route to L-ornithine from arginine--activation, selectivity and stabilization of L-arginase.

The non-proteinogenic amino acid L-ornithine (L-Orn) can be conveniently obtained by enzymatic hydrolysis of arginine (Arg) with arginase (EC 3.5.3.1). Arginase from calf liver (Vmax = 459 mumol/(min.mg), Km = 25.5 mM) is inhibited competitively by L-Orn (Ki = 480 mM). The enzyme was found to be completely enantioselective (E-value > 100) so that D,L-Arg can be split into D-Arg and L-Orn. Operational stability at 25 degrees C (deactivation rate constant kdeact = 3.8 x 10(-3) h-1; tau 1/2 = 182 h) is sufficient for use in a continuous process but is significantly smaller than temperature stability (kdeact = 4.1 x 10(-4) h-1; tau 1/2 = 1682 h); mechanical stress through stirring and unsteady Mn2+ supply owing to oxidation in the continuous process are believed to cause the difference. Addition of ascorbic acid stabilizes calf liver arginase at temperatures higher than 25 degrees C (at 60 degrees C, delta delta G not equal to = 2.9 kJ/mol).

Animals↗

PubMed2005

Probing the role of the hyper-reactive histidine residue of arginase.

Rat liver arginase (arginase I) is potently inactivated by diethyl pyrocarbonate, with a second-order rate constant of 113M(-1)s(-1) for the inactivation process at pH 7.0, 25 degrees C. Partial protection from inactivation is provided by the product of the reaction, l-ornithine, while nearly complete protection is afforded by the inhibitor pair, l-ornithine and borate. The role of H141 has been probed by mutagenesis, chemical modulation, and X-ray diffraction. The hyper-reactivity of H141 towards diethyl pyrocarbonate can be explained by its proximity to E277. A proton shuttling role for H141 is supported by its conformational mobility observed among the known arginase structures. H141 is proposed to serve as an acid/base catalyst, deprotonating the metal-bridging water molecule to generate the metal-bridging hydroxide nucleophile, and by protonating the amino group of the product to facilitate its departure.

Animals↗

PubMed2006

Ornithine transcarbamylase and arginase I deficiency are responsible for diminished urea cycle function in the human hepatoblastoma cell line HepG2.

A possible cell source for a bio-artificial liver is the human hepatblastoma-derived cell line HepG2 as it confers many hepatocyte functions, however, the urea cycle is not maintained resulting in the lack of ammonia detoxification via this cycle. We investigated urea cycle activity in HepG2 cells at both a molecular and biochemical level to determine the causes for the lack of urea cycle expression, and subsequently addressed reinstatement of the cycle by gene transfer. Metabolic labelling studies showed that urea production from 15N-ammonium chloride was not detectable in HepG2 conditioned medium, nor could 14C-labelled urea cycle intermediates be detected. Gene expression data from HepG2 cells revealed that although expression of three urea cycle genes Carbamoyl Phosphate Synthase I, Arginosuccinate Synthetase and Arginosuccinate Lyase was evident, Ornithine Transcarbamylase and Arginase I expression were completely absent. These results were confirmed by Western blot for arginase I, where no protein was detected. Radiolabelled enzyme assays showed that Ornithine Transcarbamylase functional activity was missing but that Carbamoyl Phosphate Synthase I, Arginosuccinate Synthetase and Arginosuccinate Lyase were functionally expressed at levels comparable to cultured primary human hepatocytes. To restore the urea cycle, HepG2 cells were transfected with full length Ornithine Transcarbamylase and Arginase I cDNA constructs under a CMV promoter. Co-transfected HepG2 cells displayed complete urea cycle activity, producing both labelled urea and urea cycle intermediates. This strategy could provide a cell source capable of urea synthesis, and hence ammonia detoxificatory function, which would be useful in a bio-artificial liver.

Arginase↗

PubMed2005

Spermine increases arginase activity in the liver after carbon tetrachloride-induced hepatic injury in Long-Evans rats.

Arginase is the enzyme which synthesizes urea and ornithine, a precursor from which putrescine, spermidine and spermine are formed. These natural polyamines have been implicated in cell growth, replication and wound healing. The present study evaluated the possibility that spermine increases arginase activity and reduces liver damage caused by carbon tetrachloride. Intraperitoneally injected spermine at a dose of 1 mg/kg after a single intragastric administration of carbon tetrachloride (1.6 ml/kg) increased arginase activity (6.30-7.79 microg urea/mg protein per min) (P<0.05) as well as total protein content (0.29-0.37 mg/mg dry weight) in hepatic tissue, compared to the group which only received carbon tetrachloride. When liver cell damage was biochemically assessed, the carbon tetrachloride-treated group showed a 20-fold increase in serum glutamic oxaloacetate transaminase, compared to the control group (P<0.05), and this was significantly diminished by the administration of spermine (P<0.05). Serum triglycerides increased four times compared to the control group as a result of the carbon tetrachloride treatment and were diminished by spermine as well. These results indicate that spermine may play a role in the recovery of liver tissue after carbon tetrachloride-induced liver injury, maybe by increasing the synthesis of putrescine, a polyamine which has been found out to participate in the recovery of the hepatic tissue after an insult with carbon tetrachloride.

Animals↗

PubMed2005

Synthesis and evaluation of new omega-borono-alpha-amino acids as rat liver arginase inhibitors.

Recent studies have demonstrated that arginase plays important roles in pathologies such as asthma or erectile dysfunctions. We have synthesized new omega-borono-alpha-amino acids that are analogues of the previously known arginase inhibitors S-(2-boronoethyl)-l-cysteine (BEC) and 2-amino-6-boronohexanoic acid (ABH) and evaluated them as inhibitors of purified rat liver arginase (RLA). In addition to the distance between the B(OH)(2) and the alpha-amino acid functions, the position of the sulfur atom in the side chain also appears as a key determinant for the interaction with the active site of RLA. Furthermore, substitution of the alkyl side chain of BEC by methyl groups and conformational restriction of ABH by incorporation of its side chain in a phenyl ring led to inactive compounds. These results suggest that subtle interactions govern the affinity of inhibitors for the active site of RLA.

Amino Acids↗