Specific Activity Of Arginase Research Articles

Study the characteristics of partially purified arginase from the serum of patients with diabetes mellitus type two (DMT2) in the Nineveh Governorate

Arginase plays an essential role in diabetes mellitus type 2 (DMT2). The study aimed to isolate and partially purify the serum arginase from patients with diabetes mellitus type 2, estimate its molecular weight, and determine the optimal conditions for the enzyme's action. The study included 35 patients of both sexes of 16 males and 19 females. Three purification steps were used to purify arginase are the precipitation of the protein with ammonium sulfate at a saturation of 65%, DEAE-cellulose ion-exchange chromatography, and Sephadex G-100 gel scattering technique. The results showed that the specific activity of unpurified arginine is equal to 0.6 and the specific activity increased 19 times when purified while the specific activity was equal to 11.4 (U/mg). The number of purification times was 19-fold, and recovery of 59.2 %. And the molecular weight of purified arginase equals 96050 ± 1414.2 Daltons. The highest activity of purified arginase in 250 µL serum as a source of enzyme,100 mM of buffer solution sodium barbitone, pH = 9.5, and time incubation for 45 minutes at 37 C, and 200 mM of arginine as a substrate for arginase. The study concluded that the specific activity of arginase from serum of diabetic patients increased 19 times after purification. The properties of arginine and the optimal conditions for its action are differ according to the source of the enzyme purified that from it.

Transcriptional profiling reveals molecular basis and the role of arginine in response to low-pH stress in Pichia kudriavzevii

The non-conventional yeast Pichia kudriavzevii is considered to be a promising biotechnological host for the production of organic acids under low-pH conditions. However, little is known about the low-pH stress response in P.kudriavzevii, which significantly restricts its future development. In this study, P.kudriavzevii N-X showed great tolerance to low-pH stress, but the cell aggregation upon extremely acidic conditions might be unfavorable for low-pH fermentation. We therefore conducted RNA-Seq to compare global gene expression of P.kudriavzevii N-X in response to different pH stresses. Totally 434 genes were identified to be differentially expressed genes (DEGs), and annotation and enrichment analysis suggested that multiple genes associated with regulation of membrane lipid composition, filamentous growth and arginine metabolism were differentially expressed. The increased specific activity of arginase and intracellular ammonia concentration of P.kudriavzevii cultured at pH 2.0 further implied potential roles of arginine in response to extreme low-pH conditions. Extracellular supplementation of 5mM arginine resulted in increased pHi and cell growth at pH 2.0, meanwhile the cell aggregation was partially suppressed. Additionally, overexpression of ARG J involving in arginine synthesis can also enhance the cell growth and reduce the aggregation effect. These results suggested that increasing arginine flux might be an alternative approach in the developing of P.kudriavzevii as a platform host for production of organic acids under low-pH conditions.

CAR1 deletion by CRISPR/Cas9 reduces formation of ethyl carbamate from ethanol fermentation by Saccharomyces cerevisiae.

Enormous advances in genome editing technology have been achieved in recent decades. Among newly born genome editing technologies, CRISPR/Cas9 is considered revolutionary because it is easy to use and highly precise for editing genes in target organisms. CRISPR/Cas9 technology has also been applied for removing unfavorable target genes. In this study, we used CRISPR/Cas9 technology to reduce ethyl carbamate (EC), a potential carcinogen, which was formed during the ethanol fermentation process by yeast. Because the yeast CAR1 gene encoding arginase is the key gene to form ethyl carbamate, we inactivated the yeast CAR1 gene by the complete deletion of the gene or the introduction of a nonsense mutation in the CAR1 locus using CRISPR/Cas9 technology. The engineered yeast strain showed a 98% decrease in specific activity of arginase while displaying a comparable ethanol fermentation performance. In addition, the CAR1-inactivated mutants showed reduced formation of EC and urea, as compared to the parental yeast strain. Importantly, CRISPR/Cas9 technology enabled generation of a CAR1-inactivated yeast strains without leaving remnants of heterologous genes from a vector, suggesting that the engineered yeast by CRISPR/Cas9 technology might sidestep GMO regulation.

Reduced production of ethyl carbamate for wine fermentation by deleting CAR1 in Saccharomyces cerevisiae.

Ethyl carbamate (EC), a pluripotent carcinogen, is mainly formed by a spontaneous chemical reaction of ethanol with urea in wine. The arginine, one of the major amino acids in grape musts, is metabolized by arginase (encoded by CAR1) to ornithine and urea. To reduce the production of urea and EC, an arginase-deficient recombinant strain YZ22 (Δcarl/Δcarl) was constructed from a diploid wine yeast, WY1, by successive deletion of two CAR1 alleles to block the pathway of urea production. The RT-qPCR results indicated that the YZ22 almost did not express CAR1 gene and the specific arginase activity of strain YZ22 was 12.64 times lower than that of parent strain WY1. The fermentation results showed that the content of urea and EC in wine decreased by 77.89 and 73.78 %, respectively. Furthermore, EC was forming in a much lower speed with the lower urea during wine storage. Moreover, the two CAR1 allele deletion strain YZ22 was substantially equivalent to parental strain in terms of growth and fermentation characteristics. Our research also suggested that EC in wine originates mainly from urea that is produced by the arginine.

Arginase from Bacillus thuringiensis SK 20.001: Purification, characteristics, and implications for l-ornithine biosynthesis

An l-ornithine high producing strain Bacillus thuringiensis SK20.001 was screened by our laboratory. An intracellular arginase used to biosynthesize l-ornithine from the strain was purified and characterized. The final specific arginase activity was 589.2units/mg, with 70.1 fold enrichment and 22.4% recovery. The molecular weight of the enzyme was approximately 33,000Da as evaluated by SDS-PAGE and 191,000Da as determined by gel filtration. The enzyme had an optimum pH of 10.0 and an optimum temperature of 40°C. It was stable from pH 8.0–12.0 and <50°C without Mn2+. The presence of Mn2+ and Ni2+ had strong effects on the enzyme activity, and Mn2+ significantly increased the thermal stability of the enzyme. The arginase was slightly inhibited by Ca2+, Fe2+ and Zn2+. Trp, Asp, Glu, Tyr, and Arg residues were directly involved in the arginase activity evaluated by chemical modifications. The Km and Vmax for l-arginine were estimated to be 15.6mM and 538.9μmol/min/mg. The biosynthesis yield of l-ornithine was 72.7g/L with the enzyme.

The effect of melatonin treatment on oxidative and nitrosative stress in rats with thioacetamide-induced hepatic damage

: The following study aimed to clarify the importance of arginase and NOS activities in thioacetamide-induced hepatic damage and to evaluate the underlying mechanism of proposed protection provided by melatonin, using commonly applied therapeutic dose. : Rats were randomly assigned to four groups (n=5): control, melatonin (10mg/kg i.p.), thioacetamide (200mg/kg i.p., two doses with a 24h interval) and thioacetamide+three doses of melatonin (10mg/kg i.p., prior- and post-treatment with a 24h interval before thioacetamide administrations) treated groups. : Thioacetamide administration caused hepatic damage creating oxidative and nitrosative stress accompanying perivenous necrosis and eosinophil infiltration. The significant elevation of total nitrite level in livers of thioacetamide treated groups reflected the activation of inducible nitric oxide synthase activity. The decrease in arginase activity indicated hepatic damage. Non-altered specific activity of arginase in the livers of thioacetamide treated groups did not overcome the elevation of NO production. Melatonin treatment did not modulate the levels/activities significantly. : Our results have indicated that nitrosative stress seems to be essentially critical in thioacetamide-induced hepatic failure in rats. Possible regulatory effect of arginase on NO production and applied dose of melatonin could not prevent hepatic damage.

Effectiveness of scaling and root planing versus modified Widman flap on nitric oxide synthase and arginase activity in patients with chronic periodontitis

Nitric oxide (NO) is synthesized from the conversion of L-arginine to L-citrulline by NO synthase (NOS). Arginase, which is an arginine-depleting enzyme, can compete with NOS for the common substrate L-arginine and thus inhibit NO production. In the present study, we aimed to examine the correlation between the arginase and NOS activity in patients with chronic periodontitis and to compare the effects of scaling and root planing and modified Widman flap procedures on enzyme activity. The study included 13 patients diagnosed with chronic periodontitis. Using a split-mouth design, the defects showing>or=7 mm of attachment loss were treated either with scaling and root planing or with modified Widman flap. Gingival biopsies from both sites were obtained at baseline and 2 months after periodontal treatment. Immunohistochemical staining was performed for evaluating NOS expression and specific arginase activity was determined spectrophotometrically. Although inflamed periodontal tissues demonstrated a strong inducible NOS (iNOS) expression at baseline, immunostaining decreased after periodontal treatment. iNOS expression intensity and the number of inflammatory cells showing iNOS expression were found to be higher in the scaling and root planing group compared to the modified Widman flap group. The specific activity of arginase was measured as 0.18+/-0.07 IU/mg protein in the modified Widman flap group and 0.25+/-0.11 IU/mg protein in the scaling and root planing group at baseline. After periodontal therapy, the enzyme level was increased to 0.68+/-0.14 IU/mg protein in the modified Widman flap and to 1.10+/-0.23 IU/mg protein in the scaling and root planing group. This study was the first report of evaluating the involvement of the arginine-NO pathway in chronic periodontitis and this might be considered to be of value in understanding the periodontal disease mechanisms.

Distribution and properties of arginase in the salivary glands of four species of laboratory mammals.

Important progress in arginine metabolism includes the discovery of widespread expression of two isoforms of arginase, arginase I and II, not only in hepatic cells but also in non-hepatic cells, and the formation of nitric oxide, a widely distributed signal-transducing molecule, from arginine by nitric oxide synthase. Possible physiological roles of arginase may therefore include regulation of nitric oxide synthesis through arginine availability for nitric oxide synthase. In this paper, arginase was investigated in the submandibular, sublingual, and parotid glands of rat, mouse, guinea pig, and rabbit. From their arginase contents, the salivary glands of these species were divided into two groups. Variable levels of arginase activity were detected in the salivary glands of mouse and rat. However, salivary glands of rabbit and guinea pig had almost no arginase activity. The presence of nitric oxide synthase has been reported in all the salivary glands used in this study. Therefore, one of the important findings was the presence of species specificity in the co-localization of arginase and nitric oxide synthase in the salivary glands of the four species. The highest specific activity of arginase was found in mouse parotid gland. In rat, considerable arginase activity was detected in all three glands, at 3.6-7.3% of that in rat liver. In rat submandibular gland, arginase was detected in both cytosolic and particulate fractions. In addition, arginase was detected in isolated acinar cells, but not in duct cells. Experiments on the intracellular distribution and the effects of the arginase inhibitors ornithine and N(omega)-hydroxy-L-arginine (NOHA), suggested the presence of both arginase I and arginase II in rat submandibular gland.

Significance of arginase and ornithine in malignant tumors of the human skin

During neoplastic development, several aspects of the regulation of polyamine synthesis undergo profound changes. In extrahepatic mammalian tissues in which the urea cycle is not functioning, arginase is believed to supply the cell with ornithine, a non-protein amino acid that is a precursor for biosynthesis of polyamines. Because the activity of ornithine decarboxylase and polyamine levels have been shown to be elevated during carcinogenesis, we decided to investigate the role of arginase in the development of malignant tumors of the human skin and to examine whether arginase activity and ornithine level can be used as biologic markers for distinguishing patients with squamous cell cancer from patients with basal cell cancer. For this purpose, we studied tissue arginase activity and ornithine level in tumor and adjacent normal tissues in 16 patients (55 ± 10 years of age) with malignant skin tumors (8 of which were squamous cell cancers and 8 of which were basal cell cancers). The mean arginase activity and ornithine levels in tumor tissues (total) were 17.75 ± 8.54 U/mg protein and 40.89 ± 14.88 nmol/mg protein, respectively, versus 3.69 ± 1.71 U/mg protein and 12.98 ± 6.21 nmol/mg protein, respectively, for normal tissues. The mean specific arginase activity levels in squamous cell and basal cell cancers of the human skin were 18.49 ± 10.47 U/mg protein and 16.63 ± 6.00 U/mg protein, respectively. The mean ornithine levels in squamous cell and basal cell cancers of the human skin were 42.45 ± 19.10 nmol/mg protein and 39.33 ± 10.19 nmol/mg protein, respectively. Our results indicated that (1) arginase activity and ornithine levels are elevated in squamous cell and basal cell cancers of the human skin; (2) the increased activity of arginase and hence the elevated levels of ornithine may be important in the development of malignant tumors of the human skin; and (3) although arginase activity and ornithine level may be useful for distinguishing patients with malignant skin tumors from healthy subjects, they cannot be used as biologic markers for distinguishing patients with squamous cell cancer from patients with basal cell cancer. (J Lab Clin Med 2001;137:340-4)

Arginase distribution in tissues of domestic animals

1. 1. A new colorimetric method was used for determination of arginase in different tissues of some domestic animals. 2. 2. In all species studied liver was the richest source of arginase. 3. 3. Significant differences were observed in the specific activity of arginase in livers from different species. 4. 4. In all species, besides liver, kidney and brain also contained significant levels of arginase. 5. 5. In the dog, in addition to the three organs mentioned above, lung, heart, spleen and skeletal muscle showed some arginase activity. 6. 6. In sheep and cattle significant arginase activity was observed in the rumen. No differences were observed between epithelial and muscular layers of 7. 7. These results are discussed in terms of the possible role of arginase in different tissues of animals.

Determination of argininosuccinate lyase and arginase activities with an amino acid analyzer

The measurement of argininosuccinate lyase (ASase) and arginase, both in liver and erythrocytes, was developed by using a commercial amino acid analyzer. The method is based upon the use of two different substrates, argininosuccinate and arginine for ASase and arginase, respectively, and the measurement of only one final metabolite: ornithine. The use of ornithine as a marker of biological activity of ASase is related to the fact that in the urea cycle, the specific activity of arginase is much higher than that of ASase; thus, during in vitro determinations, arginine, which is the product of ASase, is rapidly converted to ornithine. The sensitivity of the methods is very high since we were able to detect both activities using very diluted rat liver homogenates (0.10 mg protein/ml) or few microliters of human blood. In rat liver the V max for ASase and arginase were respectively 0.54 and 140 μmol/h/mg protein; the apparent K m values 1.25 and 13.5 m m. In human erythrocytes the V max for the same enzymes were 7.2 and 170 nmol/h/mg Hb and the apparent K m values were 0.66 and 9.5 m m. In 10 healthy volunteers the specific activity of ASase and arginase determined in blood were respectively 8.60 ± 0.46 and 124.1 ± 14.5 nmol/h/mg Hb. The results obtained from 2 patients suffering from argininosuccinic aciduria were also reported. In these latter cases while ASase was not detectable in blood, arginase activity was at the lowest end of the confidence limits determined in healthy volunteers.

Arginase activity in the silkworm,Bombyx mori: developmental profiles, tissue distribution and physiological role

Arginase activity was studied in the silk-worm,Bombyx mori. 1. The specific activity remained low from the larval to mid-pupal stage in both sexes. In males, it then increased markedly in the late-pupal stage, reached a maximum in young adults, and then gradually decreased with age, whereas in females it remained almost constant throughout development and ageing (Fig. 1). 2. Of all organs of both sexes, the vesicula seminalis of adult males had the highest arginase activity, the activity in this organ amounting to 95% of the total activity in adult males. The specific arginase activity in this organ was at least 50 times those in other organs. The enzyme activity in the vesicula seminalis increased very greatly during metamorphosis to adults (Tables 1, 2, 3). 3. When the vesicula seminalis was incubated withl-[14C(U)]-ornithine, large amounts of radio-activity were found in the metabolites proline and glutamate, suggesting the conversion of ornithine, produced by arginase, to proline (Fig. 2, Table 4). 4. Proline and succinate were effective substrates for the respiration of silkworm spermatozoa, whereas glucose, α-glycerophosphate and pyruvate were not used.

Activities of glutamine synthetase, glutaminase and arginase in kainic acid lesioned rat brain corpus striatum

Intrastriatal kainic acid (2 μg/μl) administration gave rise to significant increase in activities of glutamine synthetase and arginase along with a significant decrease in the activity of glutaminase in the lesioned striatal tissue 7 days after the administration of kainic acid. The increase in the activity of glutamine synthetase was attributed to the gliosis occurring in such lesions. The decrease in the activity of glutaminase was thought to be due to the loss of GABAergic neurons. The increase in arginase activity might be occurring in glial cells or in nerve endings. Although the earlier results indicated a low specific activity of arginase in glial cells, the observed increase in its activity might be partly due to its increase in proliferating glial cells, liberating ornithine for the formation of polyamines. However, it was also thought that a substantial increase may be occurring in the arginase present in the intact glutamatergic (corticostriate pathway) nerve endings, since it was earlier found that the synaptosomes of the rat brain had appreciably high activity of arginase. These results were discussed in relation to the probable roles of arginine and glutamine as the precursors for neurotransmitter pools of glutamate in striatum.

763 EVIDENCE FOR TWO GENES ENCODING HUMAN ARGINASE

Patients missing liver arginase retain considerable capacity to synthesize urea. The mechanism by which this occurs is unknown and previous biochemical and immunologic studies did not distinguish a one from a two-locus model. A kidney biopsy was obtained from M.U., an 11-year-old boy with arginase deficiency. The kidney had a specific activity of 1.34 μmol/30min/mg protein as compared to 0.35-0.87 μmol/30min/mg protein in control biopsy and autopsy kidney. When tested with rabbit anti-human liver arginase (RAHLA), the M.U. kidney demonstrated a line of identity with normal human liver and kidney. RAHLA did not precipitate kidney arginase in M.U., but did precipitate 50% of normal kidney and 100% of normal liver arginase. Precipitation-inhibition experiments demonstrated an antigenic but enzymatically inactive protein in M.U. kidney that cross-reacted with liver arginase. The data support the existence of two arginase genes in man, one expressed in liver (AI) and two in kidney (AI and AII). AI codes for enzyme active in normal liver and kidney, but not in M.U. kidney. M.U. has an inactive AI protein as demonstrated by precipitation-inhibition. All is expressed in normal kidney and in M.U. kidney. All probably arose as a result of gene duplication and appears to be inducible as demonstrated by the high specific activity of arginase in M.U. kidney. The presence of a second arginase gene expressed in kidney explains the ability of arginase-deficient patients to synthesize urea.

Arginase, an s-phase enzyme in a human cell line

Suspension cultures of ‘Chang liver’ cells were synchronized by preincubation in a glutamine-deficient medium or by thymidine blockade. Specific arginase activity varied in the synchronized cultures, being high when the number of S-phase cells was maximal. A relationship between high arginase activity and a high percentage of (S+G 2) cells was also found when unsynchronized cells were separated by velocity sedimentation. The increase in arginase activity near the G 1/S border was totally inhibited in the presence of cycloheximide. The rate of decrease in activity after addition of the drug indicated that the variations in the rate of synthesis of the enzyme, while the rate of degradation was more or less constant, corresponding to 4–6% per h. The role of arginase in cells lacking a urea cycle and the regulation of arginase activity in ‘Chang liver’ cells is discussed.

Studies on enzymes of nitrogen metabolism, and nitrogen end products in the developing chick ( Gallus domesticus) embryo

1. 1. A total of 450 fertilized eggs were used to study the concentrations of uric acid, urea and ammonia in allantoic and amniotic fluids, and some enzymes of nitrogen metabolism in the liver and kidney during the development of the chick embryo from the 5th to 21st day of incubation. 2. 2. Concentrations of the compounds studied were higher in allantoic fluid. The molar concentration of allantoic uric acid increased steadily with time. The pattern of urea and ammonia in both allantoic and amniotic fluids were the same. 3. 3. Arginase (E.C.3.5.3.1) activity in both embryonic kidney and definitive kidney was higher than that in the liver. The specific activity of arginase (μmole urea formed/hr per g wet wt kidney) dropped during development. 4. 4. Little arginine synthetase activity (argininosuccinate synthetase, E.C.6.3.4.5; and argininosuccinate lyase, E.C.4.3.2.1) was found in kidney, but non in the liver. 5. The complete urea cycle function was absent in both the liver and the kidney of the chick embryo.

The arginase and gamma-guanidinobutyrateureahydrolase of rat brain.

Summary. The synthesis of urea from y-guanidinobutyric acid (GGBA) and arginine and the possible distribution of arginase and γ-guanidinoureahydrolase has been investigated. The enzyme splitting GGBA in brain has not been described before. Arginase, as well γ-guanidinoureahydrolase, is fairly thermorésistant. The specific activity of arginase in proteins soluble in 50% acetone is 5.4 times more and of γ-guanidinoureahydrolase 5.5 times more than that in the initial homogenate. The investigation has proved our conception of the existence of the last two ways of urea formation in brain.

Repression of arginase synthesis in Chang liver cells

A rapid increase in specific arginase activity occurred in Chang liver cells immediately after addition of the missing nutrient to cells preincubated in a medium from which one essential amino acid had been omitted. The rapid change in enzymic activity offered an opportunity for studying specific enzyme regulation during short time experiments. 1. 1. The experimental results indicate that the increase in specific arginase activity, which could be immediately stopped by the addition of puromycin, was due to a synthesis of the enzyme more rapid than in normally growing cells. 2. 2. Actinomycin D at concentrations sufficient to inhibit DNA directed RNA synthesis did not prevent the increase in arginase activity, suggesting that the synthesis of the enzyme took place in response to a preformed stable messenger RNA. 3. 3. A few hours after the addition of the missing nutrient to a prestarved culture, arginase synthesis was again retarded. 3.1. (a) This decrease in the rate of enzyme formation could be prevented by addition of actinomycin D. The decrease in the rate of enzyme synthesis in absence of actinomycin, therefore, might not be due to a destruction of the specific messenger RNA, but rather to a depression of the rate at which the stable arginase messenger was translated. 3.2. (b) When proline was added to the culture medium the decrease in the rate of enzyme formation was enhanced. The experimental results suggest a regulation of arginase synthesis on the level of translation by means of an actinomycin sensitive repressor. The activity of this repressor seems to depend on the presence of a low molecular co-repressor, which accumulates in the cells in presence of proline. 4. 4. When proline was added to the deficient medium during the starvation period, the increase in arginase activity immediately after the addition of the missing nutrient was partially prevented. 4.1. (a) An increased rate of arginase formation was produced when these cells, prestarved in presence of proline, were transferred to a “normal” growth medium without added proline. 4.2. (b) This stimulation of arginase synthesis, brought about by the removal of proline, was not prevented by the addition of actinomycin D and therefore seemed not to be dependent of a formation of new messenger RNA. These results again suggest a proline dependent repression of arginase synthesis at the level of translation of a preformed, stable messenger RNA.