Arginase Reaction Research Articles

Development of a new nano arginase HPLC capillary column for the fast screening of arginase inhibitors and evaluation of their binding affinity

A simple and rapid Nano LC method has been developed for the screening of arginase inhibitors. The method is based on the immobilization of biotinylated arginase on a neutravidin functionalized nano HPLC capillary column. The arginase immobilization step performed by frontal analysis is very fast and only takes a few minutes. The miniaturized capillary column of 170 nL (length 5 cm, internal diameter 75 μm) significantly decreased the required amount of used enzyme (25 pmol). This was of significance importance when working with less available or expensive purified enzyme. Non-selective adsorption of the organic monolith matrix was reduced (<6%) and the arginase efficient yield was high (92%). The resultant affinity capillary columns showed excellent repeatability and long lifetime. The arginase reaction product was achieved within 60 s and the immobilized arginase retained 97% of the initial activity beyond 90 days. This novel approach can thus be used for the fast evaluation of recognition assay induced bya series of inhibitor molecules (caffeic acid phenylamide, chlorogenic acid, piceatannol, nor-NOHA acetate) and plant extracts.

The kinetic properties of arginase in sperm cells of inferile men

Nowadays the role of NO in the development of male infertility is actively studied. Arginase (EC 3.5.3.1) is a manganese metalloenzyme which converts L-arginine to L-ornithine and urea and reciprocally regulates NO production. Although arginase activity has usually been detected in the reproductive tract, including spermatozoa, no data relating to the kinetic properties of the enzyme in ejaculated spermatozoa has been reported. This study was designed to study the kinetic parameters of arginase of spermatozoa of infertile men. Spermatozoa arginase activity was measured by determining levels of urea production. Kinetic analysis of the enzyme reaction was performed in a standard incubation system with modified physical and chemical characteristics or the respective components (the substrate concentration, Mn2+ concentration, incubation time and protein content). Pathobiochemical and kinetic properties of sperm arginase obtained from human normozoo- and pathospermic samples were compared. The maximum rate of L-arginine hydrolysis (detrermined by L-arginine) for arginase of spermatozoa obtained from men with preserved fertility was 2.0, 1.8 and 1.9 times greater than this value for oligo-, astheno- and oligoasthenozoospermic samples respectively. However, affinity constants for L-arginine was not significantly different between fertile and infertile men. The maximum rate of L-arginine hydrolysis (deretmined by Mn2+) for arginase of spermatozoa obtained from men with preserved fertility was 1.6, 1.7 and 1.7 times greater than this value for oligo-, astheno- and oligoasthenozoospermic samples respectively. However, affinity constants for Mn2+ were not significantly different between fertile and infertile men. In the whole range of time, the urea production by arginase in sperm cells obtained from oligozoospermic samples is much lower compared to value in healthy donors. The results of kinetic analysis indicate that urea production by arginase is much more intense in the control group than in patients with various forms of pathospermia. The initial (instantaneous) reaction rate of arginase reaction was lower for oligozoospermic samples compared to normozoospermic samples. It has been found that inhibition of arginase activity in sperm cells of infertile men occurs by non-competitive type and was related to marked decrease in maximum reaction rate while affinity of arginase to L-arginine and Mn2+ was unaffected.

Arginase activity, urea, and hydroxyproline concentration are reduced in keratoconus keratocytes.

Keratoconus (KC) is a disease characterized by thinning and deformation of the cornea, but its etiology remains unknown. Seventy percent of the corneal stroma consists of collagen, which is composed of three intertwined polypeptide chains with glycine-hydroxyproline-proline repeats along their sequence. Arginase is a cytoplasmatic enzyme and catalyzes the conversion of arginine to urea and ornithine, which serves as a precursor for the endogenous synthesis of proline and hydroxyproline. The purpose of this study was to analyze arginase activity, as well as collagen and urea formation in normal and KC-keratocytes and to determine the impact of urea on keratocyte viability and proliferation in vitro. Primary human keratocytes were isolated by digestion in collagenase (1.0mg/mL) from surgically removed corneas of eight keratoconus patients and eight normal human corneal buttons and cultured in DMEM/Ham's F12 medium supplemented with 5% fetal calf serum. Arginase activity and urea concentration were measured in cell-lysates, hydroxyproline concentration in supernatant of cultured keratocytes using colorimetric assay. Cell viability and cell proliferation of cultured keratocytes were assessed after treatment with urea at concentrations up to10mM for 24h using assays for metabolic activity and DNA replication. Arginase activity and urea concentration in KC-keratocytes decreased by about 50% compared to normal keratocytes (p = 0.003 and p = 0.008). Hydroxyproline synthesized by cultured KC-keratocytes was also approximately 50% less compared to normal keratocytes (p = 0.02) and this difference decreased following treatment with 5.0 or 10.0mM urea (p = 0.02; 0.03), without any change in cell viability (p > 0.09). However, the urea treatment increased modestly (by 20%) the proliferation rate of KC-keratocytes (p = 0.04; 0.04; 0.04), without any effect on normal cultured keratocytes (p > 0.09). We identified suppressed arginase activity in the metabolic program of cultured keratoconus keratocytes. The level of urea, as one product of the enzyme arginase was also decreased. This results in impaired collagen synthesis, evidenced in the culture by reduced hydroxyproline concentration. In addition, our data showed that the other product of the arginase reaction, urea supports the proliferation of KC-keratocytes, without changes in their viability. The metabolic reprogramming of keratoconus keratocytes and its impact on development of a clinically detectable keratoconus disease has to be further analyzed.

An alternative, arginase‐independent pathway for arginine metabolism in Kluyveromyces lactis involves guanidinobutyrase as a key enzyme

Most available knowledge on fungal arginine metabolism is derived from studies on Saccharomyces cerevisiae, in which arginine catabolism is initiated by releasing urea via the arginase reaction. Orthologues of the S. cerevisiae genes encoding the first three enzymes in the arginase pathway were cloned from Kluyveromyces lactis and shown to functionally complement the corresponding deletion in S. cerevisiae. Surprisingly, deletion of the single K. lactis arginase gene KlCAR1 did not completely abolish growth on arginine as nitrogen source. Growth rate of the deletion mutant strongly increased during serial transfer in shake-flask cultures. A combination of RNAseq-based transcriptome analysis and 13C-15N-based flux analysis was used to elucidate the arginase-independent pathway. Isotopic 13C15N-enrichment in γ-aminobutyrate revealed succinate as the entry point in the TCA cycle of the alternative pathway. Transcript analysis combined with enzyme activity measurements indicated increased expression in the Klcar1Δ mutant of a guanidinobutyrase (EC.3.5.3.7), a key enzyme in a new pathway for arginine degradation. Expression of the K. lactis KLLA0F27995g (renamed KlGBU1) encoding guanidinobutyrase enabled S. cerevisiae to use guanidinobutyrate as sole nitrogen source and its deletion in K. lactis almost completely abolish growth on this nitrogen source. Phylogenetic analysis suggests that this enzyme activity is widespread in fungi.

Arginase/nitric oxide synthase regulatory mechanism of cells under the action of trigger factors that cause reactive arthritis

On the basis of the research it can be argued that the determination of the enzymatic activity of arginase and NO-synthase of peripheral blood lymphocytes gives a qualitative informative assessment on the functioning of the immune cells, as changes in the activity of arginase reflect the NO metabolism. In the research, the optimal conditions of arginase reaction were determined and some of kinetic parameters of arginase were established. The significant growth of arginase activity (3.3 times) and iNO-synthase activity (3.8 times) in peripheral blood lymphocytes in patients with reactive arthritis compared with practically healthy donors was shown.

Identification and Characterization of Proteins Involved in Rice Urea and Arginine Catabolism

Rice (Oryza sativa) production relies strongly on nitrogen (N) fertilization with urea, but the proteins involved in rice urea metabolism have not yet been characterized. Coding sequences for rice arginase, urease, and the urease accessory proteins D (UreD), F (UreF), and G (UreG) involved in urease activation were identified and cloned. The functionality of urease and the urease accessory proteins was demonstrated by complementing corresponding Arabidopsis (Arabidopsis thaliana) mutants and by multiple transient coexpression of the rice proteins in Nicotiana benthamiana. Secondary structure models of rice (plant) UreD and UreF proteins revealed a possible functional conservation to bacterial orthologs, especially for UreF. Using amino-terminally StrepII-tagged urease accessory proteins, an interaction between rice UreD and urease could be shown. Prokaryotic and eukaryotic urease activation complexes seem conserved despite limited protein sequence conservation for UreF and UreD. In plant metabolism, urea is generated by the arginase reaction. Rice arginase was transiently expressed as a carboxyl-terminally StrepII-tagged fusion protein in N. benthamiana, purified, and biochemically characterized (K(m) = 67 mm, k(cat) = 490 s(-1)). The activity depended on the presence of manganese (K(d) = 1.3 microm). In physiological experiments, urease and arginase activities were not influenced by the external N source, but sole urea nutrition imbalanced the plant amino acid profile, leading to the accumulation of asparagine and glutamine in the roots. Our data indicate that reduced plant performance with urea as N source is not a direct result of insufficient urea metabolism but may in part be caused by an imbalance of N distribution.

Der epidermale Arginin-Stoffwechsel und dessen therapeutische Beeinflussung

From a cosmetic and dermatological point of view, arginine, a physiological amino acid, is a promising active substance for topical applications. Arginine is metabolized enzymatically in the arginase reaction to urea and ornithine and in the NO-synthase reaction to NO and citrullin. Urea increases the water binding capacity of the horny skin layer, possesses keratoplastic, bacteriostatic, antimycotic, and proteolytic properties and relieves itching. NO is essentially involved in the regulation of hemo- and lymphovascular perfusion and in initiation and maintenance of inflammatory processes. It possesses immunomodulating and radical trapping properties. Both arginase and NO synthase are expressed in keratinocytes, and also in other cutaneous cells (such as melanocytes, fibroblasts, microvascular endothelial cells) and its activity is functionally influenced by various factors. In relevant concentrations, arginine can be considered safe from a toxicological point of view. As a hydrophilic substance, L-arginine can be easily worked into standard vehicle systems for topical application. L-arginine penetrates into human skin, whereby an adequate concentration time-profile is attained in the living epidermis. The potency of practical use of topically applied L-arginine arises from the biochemical role of arginine in epidermal metabolism. In addition to cosmetic use and therapy of chronic dermatoses, a number of additional applications are possible in functional and structural disruptions of the skin.

Decreased expression of arginase II in the kidneys of Dahl salt-sensitive rats.

Arginase catalyzes the hydrolysis of arginine to urea and ornithine. Urea is not only an important solute for concentrating urine but also inhibits Na-K-2Cl cotransport. To elucidate the roles of arginase in the development of salt-sensitive hypertension, we examined arginase activity and expression in the kidney and other organs of Dahl/Rapp salt-sensitive (SS) and salt-resistant (SR) rats before and after 4 weeks' administration of a 4% NaCl or control diet. At 4 weeks of age, arginase activity in the kidney was lower in SS rats than in SR rats. Kidney arginase activity was lower in SS rats than in SR rats at 8 weeks of age, and salt loading did not alter arginase activity. Arginase II (the dominant isoform in the kidney) mRNA and protein in the kidney of salt-loaded SS rats were also lower than those of salt-loaded SR rats. Arginase activities in the liver and cerebellum did not differ between SS and SR rats. To examine the effect of urea, the product of arginase reaction, on the development of hypertension, SS rats were given a 4% NaCl diet containing 5% kaolin or 5% urea. Six-week urea supplementation attenuated the development of hypertension in SS rats. These findings suggest that decreased arginase expression in the kidney may be at least partially responsible for the salt-sensitive hypertension in SS rats.

Effects of folic acid and methotrexate on arginase activity in regenerating rat liver tissue.

Arginase (L-arginine-ureidohydrolase, EC 3.5.3.1) catalyses the hydrolysis of arginine to ornithine and urea. Its primary metabolic role is to remove the excess of ammonia through synthesis of urea. Ornithine, as a product of arginase reaction can be used for synthesis of polyamines. Rapid tissue growth after partial hepatectomy is accompanied by an increase in biosynthesis of polyamines. The aim of this study was to examine the importance of arginase in regenerating liver after partial hepatectomy and to investigate the influence of folic acid and methotrexate on arginase activity on this model of compensatory growth. It was established that arginase activity was significantly reduced in regenerating rat liver tissue at different time intervals after partial hepatectomy. Administration of folic acid to hepatectomized rats led to inhibition of arginase activity. Administration of methotrexate after hepatectomy increased the arginase activity.

Possibilities and Scope of the Double Isotope Effect Method in the Elucidation of Mechanisms of Enzyme Catalyzed Reactions

Kinetic isotope effects on enzyme catalyzed reactions are indicative for the first irreversible in a sequence of individual steps. Hints on the relative velocities of other steps can only be obtained from the partitioning factor R and its dependence on external reaction conditions. In general, the experimental data needed are obtained from isotope abundance measurements in a defined position of the substrate or product as a function of turnover. This method does not reveal events dealing with neighbour atoms or preceeding the main isotope sensitive step. In the method presented here, the analytical measurement is extended to the second atom involved in a bond fission of formation (“Double Isotope Effect Method”). It is shown that the additional results obtained support the identification of the main isotopically sensitive step and its relative contribution to the overall reaction rate, the identification of other kinetically significant steps and the differentiation between stepwise and concerted reaction mechanisms. The method and its advantages are demonstrated on reactions comprising C–N-bond splitting (urease and arginase reaction), C–C-bond fission (reactions catalyzed by pyruvate-dehydrogenase, pyruvate-fonniate-lyase and lactate-oxidase), C–O-bond formation (ribulose-bisphosphate-oxygenase reaction), and N–O-bond fission (nitrate- and nitrite-reductase reactions).

Production of citrulline and ornithine by interferon-γ treated macrophages

Activated macrophages exert strong arginase (ASE) activity that converts L-arginine into ornithine, the key precursor for putrescine and polyamine biosynthesis. Macrophages were previously also shown to generate nitric oxide that is derived from the guanido group of arginine by the oxidative deiminase (OAD) reaction. In view of the physiological importance of ornithine and putrescine, we now investigated whether interferon-gamma (IFN-gamma), a principal stimulator of the OAD activity, may lead to the accumulation of the deiminated derivative citrulline at the expense of ornithine production, or whether the carbon backbone could be reutilized for the production of arginine and ornithine. Our experiments show that murine peritoneal macrophages treated with IFN-gamma in combination with tumor necrosis factor (TNF) or bacterial lipopolysaccharide (LPS) generate substantial amounts of citrulline as identified by amino acid analyzer and by thin-layer chromatography. Also, labeled citrulline is generated from [14C]L-arginine but not from [14C]L-ornithine. This suggests that macrophages have little or no capacity to convert ornithine into arginine. In the absence of IFN-gamma, TNF and LPS stimulate the conversion of arginine into ornithine but not citrulline. However, when TNF or LPS stimulated macrophages are simultaneously treated with IFN-gamma, ornithine production is relatively inhibited by the strong OAD reaction that competes with the ASE reaction for its substrate L-arginine. IFN-gamma thus down-regulates the availability of ornithine and putrescine. The lipid A precursor IA also induces, in conjunction with IFN-gamma, the production of citrulline but fails to stimulate the generation of ornithine.

Arginine Metabolism in Developing Soybean Cotyledons

Tracerkinetic experiments were performed using l-[guanidino-(14)C]arginine, l-[U-(14)C]arginine, l-[ureido-(14)C]citrulline, and l-[1-(14)C]ornithine to investigate arginine utilization in developing cotyledons of Glycine max (L.) Merrill. Excised cotyledons were injected with carrier-free (14)C compounds and incubated in sealed vials containing a CO(2) trap. The free and protein amino acids were analyzed using high performance liquid chromatography and arginine-specific enzyme-linked assays. After 4 hours, 75% and 90% of the (14)C metabolized from [guanidino-(14)C]arginine and [U-(14)C]arginine, respectively, was in protein arginine. The net protein arginine accumulation rate, calculated from the depletion of nitrogenous solutes in the cotyledon during incubation, was 17 nanomoles per cotyledon per hour. The data indicated that arginine was also catabolized by the arginase-urease reactions at a rate of 5.5 nanomoles per cotyledon per hour. Between 2 and 4 hours (14)CO(2) was also evolved from carbons other than C-6 of arginine at a rate of 11.0 nanomoles per cotyledon per hour. It is suggested that this extra (14)CO(2) was evolved during the catabolism of ornithine-derived glutamate; (14)C-ornithine was a product of the arginase reaction. A model for the estimated fluxes associated with arginine utilization in developing soybean cotyledons is presented.The maximum specific radioactivity ratios between arginine in newly synthesized protein and total free arginine in the (14)C-citrulline and (14)C-ornithine experiments indicated that only 3% of the free arginine was in the protein precursor pool, and that argininosuccinate and citrulline were present in multiple pools.

Arginaseless Neurospora: genetics, physiology, and polyamine synthesis.

Four arginaseless mutants of Neurospora crassa have been isolated. All carry mutations which lie at a single locus, aga, on linkage group VIIR. A study of aga strains shows the arginase reaction to be the major, perhaps the only, route of arginine consumption in Neurospora other than protein synthesis. Ornithine-delta-transaminase, the second enzyme of the arginine catabolic pathway, is present and normally inducible by arginine in aga strains, and ornithine transcarbamylase, an enzyme of arginine synthesis, also has normal activity. Arginine inhibits the growth of aga strains. The inhibition can be reversed by spermidine, putrescine (1,4-diaminobutane), or ornithine. The results suggest that ornithine is the major source of the putrescine moiety of polyamines in Neurospora, and that putrescine is an essential growth factor for this organism. The inhibition of aga strains by arginine can be attributed to feedback inhibition of ornithine synthesis by arginine, combined with the complete lack of ornithine normally provided by the arginase reaction.

Measurement of arginase activity

Current methods for determining arginase ( l-arginine amidinohydrolase, EC 3.5.3.1) activity involve the measurement of urea. The carbon dioxide evolved through the action of urease has been measured manometrically (1–3), although more frequently ammonia is determined by established but cumbersome procedures (4, 5). Urea also is measured colorimetrically. Its reaction with isonitrosopropriophenone in addition to requiring a one-half hour heating period leads to the formation of an unstable colored reaction product. Similarly, its reaction with xanthydrol (6, 7) results in the formation of a photolabile product, dixanthylurea. Recently a method was introduced which takes advantage of the different absorptivities of arginine, ornithine, and urea at 206 mμ (8). Urea and p-dimethylaminobenzaldehyde (Ehrlich reagent) react in dilute H 2SO 4 to form N-( p-dimethylamino)benzalurea, which is stable for a day or more. The measurement of urea in arginase reaction mixtures using this reaction is described.

Studies in arginine metabolism by schistosomes—I. Arginine uptake and lysis by Schistosoma mansoni

1. 1. In an in vivo experiment it was shown that Schistosoma mansoni worms and eggs take up C 14- l-arginine more rapidly than l-histidine, d,l-tryptophan or l-methionine. Hydrolysis of the worms and eggs indicated that worm proteins contain more arginine than do eggs. In a comparison with levels or arginine available in the host, it appears that the worms specially concentrate this amino acid in their proteins. On the other hand, eggs have a higher relative proportion of proline. 2. 2. Metabolic studies reveal that worm homogenates convert arginine to ornithine and urea. No conversion to citrulline by means of the desimidase reaction was noted. In addition, only a trace of ornithine can be produced from citrulline by ornithine transcarbamylase enzyme. It is concluded that the arginase reaction is the most important arginolytic pathway employed by this trenatode. It is highly unlikely that arginine is metabolized in order to produce ATP energy. 3. 3. The preponderance of proline in eggs is possibly linked to an arginine-ornithine-proline metabolic cycle and may be a logical explanation for the pronounced arginine uptake and lysis by schistosomes.