High Arginase Activity Research Articles

Arginase activity in different fish species and tissues

1. 1. Arginase activity was measured in different tissues from eight species of fish. 2. 2. Spur dogfish showed a very high arginase activity compared with the other species analysed. 3. 3. The activity in teleosts was mainly found in tissues of high metabolic activity (liver, kidney and red muscle).

Characterization of arginase activity from mouse epidermis and its relation to ornithine decarboxylase induction by the tumor-promoting agent, 12- O-tetradecanoylphorbol-13-acetate

Arginase, which catalyzes the cleavage of l-arginine to urea and ornithine, was detected in both soluble and particulate fractions of mouse epidermis. In a typical experiment, about 75 and 25% of the total arginase activity was associated with the soluble (100 000 × g supernatant) and the washed particulate fraction, respectively. Both soluble and particulate enzymes required the presence of divalent Mn 2+ for activity. Arginase activity was increased by about 50% in the particulate fraction, but not in the soluble fraction, by preheating the fractions at either 50 or 55°C in the presence of 15 mM MnCl 2. Enzyme activity in both fractions, in the absence of 15 mM MnCl 2, dropped precipitously during heating. A comparison of the nature of arginases in the soluble and particulate fractions revealed similar K m values (13 mM) and pH optima (9.5) and identical heat denaturation curves. Application of 10 nmol of 12- O-tetradecanoylphorbol-13-acetate to mouse skin did not increase arginase activity in either fraction over a period of 24 h. In contrast, there was a large increase in ornithine decarboxylase activity in the soluble fraction 4.5 h after treatment. Mouse epidermal ornithine decarboxylase activity was much less than arginase activity and was predominantly localized in the soluble fraction. These results indicate that the normal level of arginase activity is not a limiting factor for the stimulation of polyamine biosynthesis by TPA. High arginase activity in mouse epidermis may play a role in providing ornithine for polyamine biosynthesis and in the production of glutamate and proline as well as in the production of keratinous proteins.

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.

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.

An assay for arginase in chicken kidney

1. 1. The enzyme arginase in chicken kidney is associated with mitochondria and the mitochondrial membranes must be disrupted to obtain maximum activity. 2. 2. When the membranes were disrupted by sonication, approximately 30% higher arginase activity was observed than with the nonsonicated samples. 3. 3. The optimum pH for assay of chick kidney arginase was 9.7–9.8. Prior heat treatment with Mn 2+ decreased arginase activity. 4. 4. Highest enzyme activity was obtained by using sonicated preparations and measuring initial reaction velocity during the first 1–2 min of incubation.

Studies on functional and metabolic role of urea cycle intermediates in brain.

Abstract— The distribution of argininosuccinate synthetase, argininosuccinase and arginase, and the synthesis of urea in cerebullum. cerebral cortex and brain stem have been studied. Cerebral cortex had high levels of argininosuccinate synthetase and argininosuccinase. and a high ability to synthesize urea from aspartic acid and citrulline. Of the three regions, cerebullum had the highest arginase activity. The activities of the enzymes transamidinase and ornithine aminotransferase in the metabolism of arginine and ornithine in pathways other than urea formation have been studied in the three regions of the rat brain. The activity of creatine phosphokinase in all regions was the same: carbamylphosphatase activity was highest in cerebullum. Cerebral cortex had a high activity of aspartic acid transcarba‐mylase. The brain stem, among the three regions, had the lowest activities of glutamine synthetase and glutaminase. The activities of these enzymes in the different regions are discussed in relation to urea production and the utilization of the urea cycle intermediates.Intraperitoneal injection of high amounts of citrulline brought about a rise in the glutamine synthetase activity of cerebellum and brain stem and a rise in ornithine aminotransferase in cerebral cortex and liver. These results are discussed in relation to the mechanism of action of citrulline in alleviating the toxicity in hyperammonaemic states.

Arginase activity patterns and their regulation during embryonic development in liver and kidney

The developmental patterns for mouse liver and kidney arginase were measured by a sensitive radioactive assay from day 8 of gestation until adulthood. On day 8 high arginase activity is generally distributed throughout early embryos. Then, as development proceeds, the arginase activity drops rapidly in liver and kidney, apparently because of mass increase unaccompanied by net arginase synthesis. Suddenly, on day 12 of gestation in liver and on day 16 in kidney, arginase activity begins to accelerate toward adult values. In order to study the mechanisms controlling arginase acceleration, 12- and 13-day fetal livers were explanted to organ cultures containing various exogenous chemicals, and subsequently assayed for arginase. Physiological concentrations of hydrocortisone causes the arginase activity to rise more than 100-fold to adult levels within 4 days in culture. Glucagon, thyroxine, and dibutyryl adenosine-3′-5′-cyclic phosphate have no effect in this system. Experiments with cycloheximide, actinomycin D, and 5-fluorodeoxyuridine suggest that the hydrocortisone response is dependent upon protein and RNA synthesis but independent of DNA synthesis.

Arginase activity in erythrocytes of healthy and ill children

A quick and reliable method for the determination of blood arginase is proposed. 10 μl of blood are hemolyzed in 0.5 ml Mn 2+ and heparin solution. 25 μl of the hemolysate is activated during 20 min at 55° and thereafter incubated with arginine solution for 10 min. The reaction is then terminated by color reagent and the amount of the urea released is read, after boiling for 20 min, at 530 nm. The activity of the arginase in the young red blood cells was found to be 6% higher than in the old cells. The activity of the blood arginase of newborns was found to be 2–3-fold higher than in older children. A higher arginase activity was also found in the blood of patients with megaloblastic anemia.

THE REGULATION OF UREA-BIOSYNTHESIS ENZYMES IN VERTEBRATES.

1. Carbamoyl phosphate synthetase, ornithine transcarbamoylase, the arginine-synthetase system and arginase were measured in the livers of ammoniotelic, ureotelic and uricotelic animals. The chelonian reptiles, whose nitrogen excretory patterns vary according to the habitat, and the Mexican axolotl, a neotenic species, were also studied. 2. The levels of the activities of the first three enzymes mentioned correlate with the amount of nitrogen excreted as urea. 3. The terrestrial turtle, which excretes mainly uric acid, maintains a high arginase activity but has very low levels of the activities of the other three enzymes. 4. The first three enzymes of the urea cycle vary in the phylogenic scale in a co-ordinated manner, which suggests that they are under the same regulatory mechanism. 5. Urea formation from endogenous arginine in vitro has a low efficiency in the Mexican axolotl. 6. The induction of metamorphosis in the Mexican axolotl by the administration of l-tri-iodothyronine, which causes a shift from ammonio-ureotelism to complete ureotelism, is accompanied by an increase mainly in carbamoyl phosphate synthetase and also by an improvement in the efficiency of hydrolysis of endogenous arginine in vitro to give urea. 7. The results obtained by differential centrifugation of the urea-cycle enzymes in rat and Mexican-axolotl livers are presented. The location requirements for the integration of a metabolic cycle are discussed.

HANDLING OF UREA AND RELATED COMPOUNDS BY THE RENAL TUBULES OF THE FROG.

A high arginase activity is found in the kidney of the adult frog Rana catesbeiana which actively secretes urea. Urea accumulates in the kidney in concentrations around seven to eight times the blood concentration. To test whether the kidney arginase causes significant formation of urea in tubular cells at normal plasma arginine levels, C14 urea, chemical urea, and creatinine clearances were measured. C14 urea and chemical urea clearances were identical, both being about seven times the GFR. 2,4-Dinitrophenol (DNP) inhibited the active secretion of urea, but C14 and chemical urea clearances remained identical to each other. When arginine was administered, urea was formed in the kidney and excreted in the urine at the rate of 35 µm/g kidney hr. The chemical urea clearance was then about twice the C14 urea clearance. DNP administered with arginine caused the C14 urea clearance to fall below the GFR but did not inhibit the formation of urea as shown by the chemical urea clearance being three times the GFR. Compounds chemically related to urea were handled in different ways by the frog kidney. Acetamide and methylurea were not secreted and did not accumulate in the kidney. DNP had little or no affect on their excretion. Thiourea was found to be actively secreted and to accumulate in the kidney. DNP inhibited thiourea secretion. It is concluded that urea is normally actively transported across the tubular wall and that formation of urea in the tubular cells is insignificant.

On the substrate induced enzyme formation in animal cells cultured in vitro

The substrate induced formation of enzymes has been studied in cells cultured in vitro. Two different systems of cell cultures have been used: cell strains, established during longterm cultivation in vitro, and freshly isolated embryonic cells. In cell strains it was not possible to induce enzyme formation by the addition of substrates to alkaline or acid phosphatase, arginase, acetylcholinc esterase, d-amino acid oxidase, adenosine deaminase, asparaginase or tryptophan peroxidase, when the cells were grown either in Eagle's medium or in Parker's medium 199. In freshly isolated embryonic chick and mouse cells on the other hand, addition of arginine resulted in a higher arginase activity than in the controls if the cells were in Eagle's medium. No such response was found in Parker's medium 199. When RNA was added the arginase activity in the cells increased by a factor of about five and induction of the enzyme as response to added arginine became possible in either medium. It is suggested that the results be interpreted in terms of a competition for RNA precursors between “conventional” enzyme-synthesizing templates of the cell and “unusual” arginase-synthesizing templates.

The arginase and rhodanese activities of certain cell strains after long cultivation in vitro.

The activities of the enzymes arginase and rhodanese were examined in homogenates of 13 mouse cell strains and 2 human cell strains after long cultivation of the cells in vitro. Three strains of mouse liver origin showed both high arginase and rhodanese activities in keeping with activities of the tissue of origin. Two strains of cells of human epithelial origin, HeLa and epidermis, were found to have high rhodanese activity but low arginase activity, the skin being almost devoid of it. Seven mouse fibroblast strains or substrains had moderate rhodanese activity and all except one of the strains had low arginase activity. Two of the fibroblast strains tested, NCTC 1745 and NCTC 2050, were derived from a single cell of a tumor appearing after injection of the high tumor producing strain NCTC 1742 into a mouse. The strain 1745 had 20 times and the strain 2050 had 400 times the arginase activity of the parent strain of cells. The findings imply a considerable biochemical variation in the three strains. Three mouse mammary carcinoma clones were devoid of arginase activity, but had considerable rhodanese activity.