Bovine Liver Arginase Research Articles

Optimization of the arginase activity assay micromethod for macrophages and sera

ObjectiveWe optimized the spectrophotometric micromethod for the determination of arginase activity based on the Corraliza et al. modification of Schimke’s method. Arginase activity in sera from patients suffering from human African trypanosomiasis, in macrophage lysates from trypanosome-infected mice, and in purified bovine liver arginase was compared using the conventional and optimized micromethods.ResultsThe sensitivity of both micromethods was comparable. However, our optimized method has the following advantages: it uses small sample volumes (6 µl per assay vs. 50 µl) and reagent volumes (200 µl vs. 400 µl), it can be carried out in a single microplate well, thereby minimizing handling, and it requires fewer materials and utilizes readily available equipment. Our optimized method proved to be applicable and well suited for small-volume samples and resource-poor laboratories.

Lysine and homoarginine are closely interrelated metabolites in the rat

L-Lysine (Lys) and L-arginine (Arg), but not L-homoarginine (hArg), are proteinogenic amino acids. In healthy humans, oral administration of hArg increased the plasma concentration of Lys, suggesting Lys as a metabolite of hArg. In humans and animals, hArg is biosynthesized from Arg and Lys by arginine:glycine amidinotransferase (AGAT). In vitro, recombinant human arginase and bovine liver arginase I hydrolyzed hArg to Lys, suggesting Lys as a metabolite of hArg. The aim of the present study was to investigate whether changes in blood concentrations of hArg and Lys in old rats fed for 4 months with varied controlled experimental diets could suggest interconversion of these amino acids. Blood samples (n = 253) were taken before (T0) and after 2 months (T2) and 4 months (T4) of the experiment. Plasma concentrations of Lys and hArg were determined by gas chromatography–mass spectrometry. The plasma hArg concentration markedly correlated with the plasma Lys concentration at all timepoints (r ≥ 0.7, P < 0.0001). Further analysis demonstrated that hArg and Lys are closely and specifically associated independently of experimental time/rat age and diet, suggesting that hArg and Lys are mutual metabolites in old rats. Based on the plasma concentration changes, the median yield of hArg from Lys was determined to be 0.17% at T0 and each 0.27% at T2 and T4. With a circulating concentration of about 3 µM, hArg a major metabolite of Lys in healthy humans. hArg supplementation is currently investigated as a cardioprotective means to improve impaired hArg synthesis. Present knowledge suggests that Lys rather than hArg supplementation may be even more favorable.

P.139 Chemical models of dysfunctions of the brain 2-oxo acid dehydrogenases in vivo

l-Homoarginine (hArg) is biosynthesized from l-arginine (Arg) and l-lysine (Lys) by arginine:glycine amidinotransferase (AGAT). AGAT also catalyzes the formation of guanidinoacetate (GAA) from Arg and glycine (Gly). GAA is converted to creatine (N-methyl guanidinoacetate) by guanidinoacetate N-methyl-transferase (GAMT). Low circulating and excretory concentrations of hArg are associated with worse cardiovascular outcome and mortality. hArg is a poor substrate of nitric oxide synthase (NOS) and a weak inhibitor of arginase. The metabolism of hArg in humans is little investigated. Previously, we found that orally administered hArg (125 mg/day) increased the plasma concentration of hArg, but not of Arg, the substrate of NOS, in healthy subjects. We newly analyzed the plasma samples collected in that study for Lys and other amino acids. Repeated measures ANOVA revealed statistically significant differences between the groups (P = 0.008) with respect to plasma Lys concentration which increased by about 8% after a 4-week hArg supplementation. In vitro, recombinant human arginase and bovine liver arginase I were demonstrated by a specific and sensitive stable-isotope GC-MS assay to hydrolyze hArg to Lys. Our results suggest that Lys is a metabolite of hArg produced by the hydrolytic activity of arginase. Arginase may play a key role in hArg homeostasis in humans.

Vasorelaxant effects of Crataegus pentagyna: Links with arginase inhibition and phenolic profile

Ethnopharmacological relevanceCrataegus leaves, flowers and fruits have been traditionally used to improve blood circulation, numerous preclinical and clinical studies supporting the cardiovascular benefits of Crataegus preparations. In this respect, there is very limited data on Crataegus pentagyna; in addition, the chemical profile of this species is still incompletely elucidated. Aim of the studyThe objective of this study was to examine the cardiovascular benefits of Crataegus pentagyna Waldst. et Kit. ex Willd. (small-flowered black hawthorn, Rosaceae) extracts (leaf, flower and fruit ethyl acetate extracts) and the underlying mechanisms. We hypothesized that C. pentagyna extracts might exert vasodilatory effects and inhibit arginase activity due, in large part, to their polyphenolic constituents. Materials and methodsC. pentagyna extracts induced-relaxation and the mechanisms involved were studied ex vivo in isolated aortic rings from Sprague-Dawley rats. The inhibitory effects on bovine liver arginase I were assessed by an in vitro assay. Metabolite profiling of C. pentagyna extracts was performed and the most endothelium- and nitric oxide synthase-dependent; flower extract additionally reduced Ca2+ entry and, to a lesser extent, Ca2+ release from the sarcoplasmic reticulum. C. pentagyna proved to be an important source of arginase inhibitors with potential benefits in endothelial dysfunction that remains to be explored.

Evidence by GC-MS that lysine is an arginase-catalyzed metabolite of homoarginine in vitro and in vivo in humans

l-Homoarginine (hArg) is biosynthesized from l-arginine (Arg) and l-lysine (Lys) by arginine:glycine amidinotransferase (AGAT). AGAT also catalyzes the formation of guanidinoacetate (GAA) from Arg and glycine (Gly). GAA is converted to creatine (N-methyl guanidinoacetate) by guanidinoacetate N-methyl-transferase (GAMT). Low circulating and excretory concentrations of hArg are associated with worse cardiovascular outcome and mortality. hArg is a poor substrate of nitric oxide synthase (NOS) and a weak inhibitor of arginase. The metabolism of hArg in humans is little investigated. Previously, we found that orally administered hArg (125 mg/day) increased the plasma concentration of hArg, but not of Arg, the substrate of NOS, in healthy subjects. We newly analyzed the plasma samples collected in that study for Lys and other amino acids. Repeated measures ANOVA revealed statistically significant differences between the groups (P = 0.008) with respect to plasma Lys concentration which increased by about 8% after a 4-week hArg supplementation. In vitro, recombinant human arginase and bovine liver arginase I were demonstrated by a specific and sensitive stable-isotope GC-MS assay to hydrolyze hArg to Lys. Our results suggest that Lys is a metabolite of hArg produced by the hydrolytic activity of arginase. Arginase may play a key role in hArg homeostasis in humans.

Inhibitory Effect on Arginase and Total Phenolic Content Determination of Extracts from Different parts of Melastoma malabathricum L.

Background: Some phenolic compounds are potential source for arginase inhibitor that can be used in management of disease associated with endothelial dysfunction. Objective: The aim of this study was to investigate the inhibitory effect on arginase and to determine total phenolic content of extracts from different parts of Melastoma malabathricum L. Methods: Each part were extracted with 70% ethanol by maceration. The extracts were mixed with bovine liver arginase and L-arginine as a substrate. The inhibitory effect of extracts were determined in vitro, by measuring its absorbance on 430 nm. Total phenolic content were determined with Folin-Ciocalteu 25% on 750 nm. Results: The 70% ethanol extract of M. malabathricum leaves at 100 μg/mL was found to have highest inhibition (81.26 ± 5.27 %) followed by flower 73.39 ± 9.39%, fruit 67.63 ± 7.61 and stem 61.61 ± 4.56%. The IC50 value of the most active extract was 62.43 μg/mL while the IC50 value of N-hidroksi-nor-L-arginin (nor-NOHA) acetate, an arginase inhibitor was found to be 3.91 μg/mL. Total phenolic content of 70% ethanolic extracts of M. malabathricum leaves, flower, fruit and stem was 15.9, 20.7, 6.44, and 6.29%. Conclusion: The 70% ethanol extract of M. malabathricum leaves exhibited the highest inhibition on arginase but highest total phenolic content was from flower part. Pearson correlation test showed no correlation p>0.05 between arginase inhibition and total phenolic content of the samples. Key words: Arginase, L-arginine, Melastoma malabathricum.

Investigation of Mammal Arginase Inhibitory Properties of Natural Ubiquitous Polyphenols by Using an Optimized Colorimetric Microplate Assay.

Polyphenols are plant secondary metabolites which possess many positive effects on human health. Although these beneficial effects could be mediated through an increase in nitric oxide synthase activity, little is known regarding the inhibitory effect of polyphenols on mammal arginase, an enzyme which competes with nitric oxide synthase for their common substrate, L-arginine. The aim of the present study was to determine the potential of a series of polyphenols as mammalian arginase inhibitors and to identify some structure-activity relationships. For this purpose, we first developed a simple and cost-effective in vitro colorimetric microplate method using commercially-available mammal bovine liver arginase (b-ARG 1). Among the ten tested polyphenolic compounds [chlorogenic acid, piceatannol, resveratrol, (-)-epicatechin, taxifolin, quercetin, fisetin, caffeic acid, quinic acid, and kaempferol], cholorogenic acid and piceatannol exhibited the highest inhibitory activities (IC50 = 10.6 and 12.1 µM, respectively) but were however less active as (S)-(2-Boronoethyl)-L-cysteine (IC50 = 3.3 µM), used as reference compound. Enzyme kinetic studies showed that both chlorogenic acid and piceatannol are competitive arginase inhibitors. Structural data identified the importance of the caffeoyl (3,4-dihydroxycinnamoyl)-part and of the catechol function in the inhibitory activity of the tested compounds. These results identified chlorogenic acid and piceatannol as two potential core structures for the design of new arginase inhibitors.

Thermokinetic Studies on the Activation of Arginase by Glycine

AbstractThe activation of bovine liver arginase, which catalyzes the hydrolysis of L‐arginine to L‐ornithine and urea, by glycine was studied by thermokinetic methods at 37°C in 40 mmol·L−1 sodium barbiturate‐HCl buffer solution (pH 9.4). Results of this experiment indicate that an appropriate concentration of glycine can enhance the activity of arginase, and the relative activation rate reached its maximum value, 74%, when the concentration of glycine in reaction system was 1 mmol·L−1 and the initial concentration of arginine was 5 mmol·L−1. With the increase of substrate concentration, the relative activation rate decreased in a definite glycine concentration. Michealis constant Km of reaction decreased from 5.53 to 3.31 mmol·L−1 and inhibition constant of product L‐ornithine Kp increased from 1.18 to 3.73 mmol·L−1 when glycine concentration was 1 mmol·L−1. For these reasons one possible activation mechanism of arginase by glycine was suggested that the activation effect results from the competition of glycine and arginine to enzyme activity position. When one or two of the activity positions of arginase are occupied by glycine, it is propitious for the enzyme to complex with substrate and obstruct L‐ornithine from combining with enzyme, and when all of the activity positions are occupied by glycine, the activation effect vanishs and the inhibition effect appears.

Nδ-Methylated l-arginine derivatives and their effects on the nitric oxide generating system

So far N δ-methyl- l-arginine (MA) is only detected in yeast cells. Assuming that MA also exists in mammalians we examined possible physiological effects of N δ-methylated l-arginine derivatives on the nitric oxide generating system, that is, nitric oxide synthase (NOS), arginase and dimethylarginine dimethylaminohydrolase (DDAH). N δ-methyl- l-citrulline (MC) turned out to be a weak non-specific inhibitor of nitric oxide synthases. Moreover, MA is hydroxylated by all human NOS isoforms to N ω-hydroxy- N δ-methyl- l-arginine (NHAM) but not converted further. This hydroxylated intermediate, however, was detected to be a potent inhibitor of bovine liver arginase with a K i of 17.1 ± 2.2 μM.

Thermokinetic study on the reversible competitive inhibition of bovine liver arginase

AbstractA new thermokinetic reduced extent method for studying of the reversible Competitive inhibition of single substrate enzyme‐catalyzed reactions was proposed in this paper. The reaction that arginase‐catalyzed hydrolysis of L‐arginine to L‐ornithine and urea and the inhibition of this reaction by the product, L‐ornithine, and exogenous L‐lysine were studied at 37 °C in 40 mmol−L−1 sodium barbiturate‐HC1 buffer solution (pH=9.4). Michealis constant Km for arginine and maximum velocity Vm of the reaction were determined to be 5.14 mmol.L−1 and 1.13X 10−‐2 mmol‐L−1.s‐1, respectively. The product inhibition constant Kp and inhibitory constant K1 of L‐lysine were determined to be 1.18 and 5.6 mmol.L−1, respectively. All the results have better repeatability and self‐consistency and are in agreement with literature values. This new method using more direct thermal information from the process would give more reliable kinetic information than the traditional initial rate method.

Microcalorimetric Studies on the Product Inhibition of Hydrolysis of L-arginine Catalyzed by Bovine Liver Arginase

A new thermokinetic reduced extent method for the product inhibition of single substrate enzyme-catalyzed reactions is proposed and compared with the traditional initial rate method in this paper. The arginase-catalyzed hydrolysis of L-arginine to L-ornithine and urea was studied at 37°C in 40 mM sodium barbiturate-HCl buffer solution (pH=9.4). Michaelis constant (K m ) for arginine and maximum velocity (V m ) of the reaction were determined by initial method and thermokinetic method. The activation of exogenous manganese to this reaction was also studied. The product inhibition constant (K p ), which cannot be obtained directly from the initial rate method, was determined by thermokinetic without adding L-ornithine to the reaction system. When the concentration of Mn 2+ in cell is 0.1 mM, the enzyme gets its full activity. Incubation arginase with appropriate concentration of Mn 2+ resulted in increased V max and a higher sensitivity of the enzyme to product with no change in the K m for arginine. We suggest that the exogenous manganese ions in solution have just recovered the activity of arginase, which was lost in dissolving and dilution, but no effect on the mechanism of the reaction.

Microcalorimetric studies on the non-competitive inhibition of bovine liver arginase

Two methods, e.g. initial rate method and thermokinetic reduced extent method were presented for studies on non-competitive inhibition. Arginase-catalyzed the hydrolysis of L-arginine toL-ornithine and urea and the inhibition of this reaction by sodium fluoride were studied in the absence and presence of exogenous of Mn2+at 37°C in 40 mM sodium barbiturate-hydrochloric acid buffer solution (pH 7.4). Both methods were successively used to determine the values of K1. The advances and disadvantages of each method were compared in this paper. Exogenous Mn2+ could result in more sensitivity of arginase to F-1. Since the inhibition of arginase by F-1 depends on the pH values of the reaction system and behave as a non-competitive inhibition, it probably due to its small volume and high electronic density allow it access to the activity site of the enzyme and replaces of µ-OH2 (or µ-OH) as the bridge ligand with Mn(II, II) cluster. However, further studies are necessary to determine the modes of interaction of F-1 with bovine liver arginase.

Thermokinetic studies on the activation of bovine liver arginase by manganese ions

The activation of bovine liver arginase, which catalyzes the hydrolysis of l-arginine to l-ornithine and urea, by manganese ions was studied by thermokinetic methods at 37 °C in 40 mM sodium barbiturate–HCl buffer solution (pH 9.4). Full activation of arginase, by incubation with 0.1 mM Mn 2+, resulted in increased of V max, and a higher sensitivity of the enzyme to product and l-lysine inhibition, with no change in the K m for arginine. Upon addition of 0.1 mM Mn 2+ to the reaction, the inhibitory constants of product ( K P) and l-lysine ( K I) decreased from 1.18 to 0.70 mM and from 5.60 to 3.10 mM, respectively. We suggest that the exogenous manganese ions in reaction recovered the activity of arginase, which was lost in dissolving and dilution, without effecting on the mechanism of the reaction.

The flux of arginine after ischemia-reperfusion in the rat kidney

Renal arginine synthesis is regulated by arginine plasma levels. The amino acid arginine is synthesized in the proximal tubule of the kidney. Renal ischemia reperfusion (I-R) injury as seen after shock, trauma and major vascular surgery, leading to acute tubular necrosis, might reduce arginine production. Wistar rats received either bovine liver arginase (ASE), to lower arginine plasma levels, or saline (SAL). Following the ASE or SAL infusion, rats were randomized to receive a renal artery clamp for 70 minutes, followed by 150 minutes of reperfusion. Renal arteriovenous blood samples were measured and plasma flow was calculated in the I-R kidney (SAL/I-R and ASE/I-R) and the contralateral kidney (SAL/C-L and ASE/C-L) in order to determine renal arginine metabolism. Arginase infusion resulted in lower arginine plasma levels compared to SAL treatment (SAL/I-R vs. ASE/I-R, P < 0.005, and SAL/C-L vs. ASE/C-L, P < 0.005). Renal plasma flow was similar for all groups. The kidney switched from arginine production into arginine uptake after ischemia reperfusion (SAL/I-R vs. SAL/C-L, P < 0.01, and ASE/I-R vs. ASE/C-L, P < 0.01). Renal uptake of glutamine and citrulline increased after ischemia reperfusion (SAL/I-R vs. SAL/C-L and ASE/I-R vs. ASE/C-L, both P < 0.01). Histopathological slices of the kidney showed significantly higher counts of hyperchromasia, pyknosis, nuclear fragmentation and mitoses in individual kidney cells after ischemia reperfusion. Decreased renal arginine production is observed with unilateral ischemia-reperfusion, and this change in arginine flux could contribute to or slow the recovery from the low plasma levels of arginine seen in conditions like trauma, shock, or after vascular procedures.

Reduced arginine plasma levels are the drive for arginine production by the kidney in the rat.

In bile duct ligated rats, arginase (ASE) release from damaged hepatocytes results in low arginine (ARG) levels despite maximal renal ARG production. Plasma ARG levels were restored by reducing gut-derived endotoxemia that lowered circulating ASE activity although maintaining increased renal production. From this it was not clear if the higher renal ARG production was induced by the low grade endotoxemia or the low arginine plasma levels. The separate and combined influence of both factors on ARG metabolism was studied in the rat. Male Wistar rats received either bovine liver ASE, to lower ARG levels, or saline (SAL). Following the ASE or SAL infusion, rats were randomized to receive a low dose endotoxin (LPS) or SAL infusion. In ASE/SAL- and ASE/LPS-treated rats, ARG levels were lower compared with SAL/SAL (p<.005) and SAL/LPS (p<.005). The increased ARG production by the kidneys and gut proved to be independent of LPS but related to reduced ARG plasma levels (both p<.05 when compared with SAL/SAL and SAL/LPS). Metabolism of related amino acids was not explanatory. The study concluded that a low grade endotoxemia did not influence the metabolism of ARG by the gut, kidney, and liver. Reductions in ARG plasma by ASE treatment, irrespective a low dose endotoxin, were the drive for ARG production by the gut and the kidney.

α-Difluoromethylornithine (DFMO) as a Potent Arginase Activity Inhibitor in Human Colon Carcinoma Cells

α-Difluoromethylornithine (DFMO) is commonly used as a specific ornithine decarboxylase (ODC, EC4.1.1.17) irreversible inhibitor. ODC is the enzyme responsible for polyamine biosynthesis, which has been shown to be strictly necessary for cell proliferation. In HT-29 Glc −/+ cells, l-arginine is the major precursor of these molecules through the sequential actions of arginase, which leads to l-ornithine generation and ODC. l-ornithine, a substrate for ODC, retroinhibits arginase. Since DFMO is an ornithine analogue, we searched for a direct effect of this agent upon arginase. The flux of l-arginine through arginase in intact cells was inhibited by 51 ± 11% by 10 mM of DFMO whereas 10 mM of l-valine, a known potent arginase inhibitor, inhibited this flux by 73 ± 6%. DFMO equilibrated between extracellular and intercellular spaces and, when used at 10-mM concentration, was without effect on l-arginine net uptake. Measurement of arginase activity in HT-29 cell homogenates with increasing concentrations of DFMO and l-arginine led to an inhibition with a calculated K i (inhibitory constant) equal to 3.9 ± 1.0 mM. l-ornithine was less effective than DFMO in inhibiting arginase activity. Bovine liver arginase, used as another source of the enzyme, was also severely inhibited by DFMO. The inhibitory effect of DFMO upon arginase, one step upstream of the ODC reaction in the metabolic conversion of l-arginine to polyamines, is of potential physiological importance, since it could alter the production of ornithine and thus its metabolism in pathways other than the ODC pathway.

L-Arginine depletion by arginase reduces nitric oxide production in endotoxic shock: an electron paramagnetic resonance study

Nitric oxide (NO) synthesis was measured in the liver, lung, spleen and kidney of lipopolysaccharide-treated male rats using the nitric oxide spin trap, iron (II)-diethyldithiocarbamate (FeDETC 2). Nitric oxide formation in vivo was determined by the increase in intensity of the characteristic triplet hyperfine EPR spectrum of [NO-FeDETC 2]. Intravenous bovine liver arginase, at a dose which completely depleted circulating arginine, significantly reduced the formation of nitric oxide in these tissues. The general decrease in NO levels was confirmed by the decrease in plasma nitrite levels. These results directly demonstrate that NO formation in endotoxic shock depends on extracellular arginine; depletion of plasma arginine may be a useful therapeutic strategy.

Immobilization, characterization, and laboratory-scale application of bovine liver arginase

Arginase isolated from beef liver was covalently attached to a polyacrylamide bead support bearing carboxylic groups activated by a water-soluble carbodiimide. The most favorable carbodiimide was N-cyclohexyl-N'-(methyl-2-p-nitrophenyl-2-oxoethyl) aminopropyl carbodiimide methyl bromide, but for practical purposes, N-cyclohexyl-N'-morpholinoethyl carbodiimide methyl tosylate was used. The optimal conditions for the coupling procedure were determined. The catalytic activity of the immobilized arginase was 290-340 U/g solid or 2.9-3.4 U/mL wet gel. The pH optimum for the catalytic activity was pH 9.5, the apparent temperature maximum was at 60 degrees C and Kmapp was calculated to be 0.37M L-arginine. Immobilization markedly improved the conformational stability of arginase. At 60 degrees C, the pH for maximal stability was found to be 8.0. The immobilized arginase was used for the production of L-ornithine and D-arginine.

Nω-Hydroxy-L-Arginine, an Intermediate in the L-Arginine to Nitric Oxide Pathway, Is a Strong Inhibitor of Liver and Macrophage Arginase

Nω-Hydroxy-L-arginine (L-NOHA) is a potent inhibitor of the hydrolysis of L-arginine (L-Arg) to L-ornithine (L-Orn) catalyzed by purified bovine liver arginase (BLA). It appears as one of the most powerful arginase inhibitors reported so far (Ki = 150 μM). The other products of NO synthase are either without effect (NO2−, NO3−) or much weaker inhibitors (L-citrulline (L-Cit) and NO) of BLA. Products derived from a possible hydrolysis of L-Arg (L-Orn and urea) or of L-NOHA (L-Cit, hydroxyurea and hydroxylamine) are also inactive toward BLA at concentrations up to 2 mM. The configuration of L-NOHA is important as D-NOHA is much less active, and its free -COOH and α-NH2 functions are required for recognition of BLA. L-NOHA is also a potent inhibitor of the arginase activity of rat liver homogenates and of murine macrophages (IC50 of 150 and 450 μM, respectively). These remarkable properties of L-NOHA could play a role in the modulation of the biosynthesis of the biological mediator NO by increasing local L-Arg concentrations.

Possible involvement of manganese in the catalytic mechanism of bovine liver arginase

1. 1. Bovine liver arginase followed Michaelis-Menten kinetics in the pH range of 4.5–9.0. The variation of v i with pH implied that a basic group (p K a 8.7) functions at the catalytic site. 2. 2. Treatment of the enzyme with N-ethylmaleimide showed that there are no critical sulfhydryl groups on the enzyme. 3. 3. The less selective reagent, 3-bromopyruvate, caused biphasic inactivation which was unaffected by the presence of ornithine. 4. 4. The data pointed against critical involvement of active site amino acid side chains in the catalytic sequence in arginase. 5. 5. The observed pH-rate profile may reflect ionization of metal-bound water.