Arginase from rat fibrosarcoma. Purification and properties

Arginase from rat fibrosarcoma was purified about 1900-fold and its properties were compared with those of the enzyme from liver and kidney. Arginase from fibrosarcoma was a neutral protein of molecular weight 120,000 with a K_m value of 11 mM for arginine. The activation energy was 7.2 kcal/mol and the pH optimum was 10. The fibrosarcoma enzyme was immunologically different from that of the liver. The arginase from fibrosarcoma closely resembled the arginase from the kidney in its electrophoretic, kinetic and immunological properties.     

Purification and characterization of arginase from Neurospora crassa

We have purified an enzymatically active form of arginase from a wild-type strain of Neurospora crassa to homogeneity. The enzyme has a subunit molecular weight of 38,300 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The native protein migrated as a hexamer during gel-filtration chromatography with an apparent molecular weight of 266,000. The enzyme exhibited hyperbolic kinetics at pH 9.5 with an apparent Km for arginine of 131 mM. Antiserum was prepared against the purified enzyme and used to demonstrate the existence of three cross-reactive proteins in crude extracts of wild-type N. crassa. One of these proteins corresponded to the purified protein, whereas the other two were of molecular weights 41,700 and 26,800, respectively. Using the same antiserum, we found that rat liver, but not rat kidney, contains immunoreactive material. We also detected two proteins in extracts of Saccharomyces cerevisiae that were weakly cross-reactive with the antiserum. These data provide evidence for the existence of multiple forms of arginase in fungi as well as in mammals.     

Molecular charcteristics of chicken kidney arginase.

Chicken kidney contains two arginases with different sedimentation coefficients and substrate specificity. The ligher of these arginases, which hydrolyses only L-arginine, has been purified about 3000-fold. Like the "ureotelic" arginase, developed in chicken liver after starvation, it displays many of the properties of the arginase of the "ureotelic" species. This seems to exclude the possibility that ureotelism and uricotelism are characterized by a specific type of arginases. Both liver and kidney arginases are located in the mitochondrial matrix. The rate of hydrolysis of arginine thus not only depends on the arginase activity but also on the rate of transport of arginine into the matrix. This last process therefore is of regulatory significance.     

Assay and kinetics of arginase

A sensitive colorimetric assay for arginase was developed. Urea produced by arginase was hydrolyzed to ammonia by urease, the ammonia was converted to indophenol, and the absorbance was measured at 570 nm. The assay is useful with low concentrations of arginase (0.5 munit or less than 1 ng rat liver arginase) and with a wide range of arginine concentrations (50 microM to 12.5 mM). Michaelis-Menten kinetics and a Km for arginine of 1.7 mM were obtained for Mn2+-activated rat liver arginase; the unactivated enzyme did not display linear behavior on double-reciprocal plots. The kinetic data for unactivated arginase indicated either negative cooperativity or two types of active sites on the arginase tetramer with different affinities for arginine. The new assay is particularly well suited for kinetic studies of activated and unactivated arginase.     

Microheterogeneity of molecular forms of arginase in mammalian tissues

Two isoforms of arginase, A1 and A2, were found in rat liver, submaxillary gland and kidney as well as beef kidney. In beef liver, however, A2 was the only detectable form. Two additional forms, A3 and A4, found only in rat kidney were probably artifactitious. A1 and A2 exhibited chromatographic and immunological microheterogeneity. While A1 in rat liver and submaxillary gland was excluded by DEAE-cellulose (pH 8.3) and retained on CM-cellulose (pH 7.5), that (A'1) in beef and rat kidneys was excluded by both ion-exchangers. A2 in all tissues was retained on DEAE-cellulose, but not on CM-cellulose. Both A1 and A2 in rat liver and beef kidney, A1 from rat submaxillary gland and A2 from beef liver were precipitated by antibodies to rat and beef liver arginases. None of the forms in rat kidney (A1, A2, A3 and A4) showed any cross-reactivity to either antibody. Rat submaxillary gland A2 was precipitated by anti-rat liver arginase, but activated by anti-beef liver arginase. While the major molecular forms were A1 in rat liver and submaxillary gland and A2 in beef liver and rat kidney, the two forms occurred in equal proportions in beef kidney. It appears that different isoforms might function as components of the urea cycle in the liver of different mammals and of the arginine catabolic pathway in different extrahepatic tissues.     

Purification of rat kidney arginases A1 and A4 and their subcellular distribution

Arginase A1 and arginase A4 were isolated from rat kidney. Arginase A4, which is the main form of arginase in rat kidney, was obtained at a highly purified preparation; its specific activity was 1057 mumoles ornithine . min-1 . mg-1 protein. The two forms differed in subcellular localization. Form A1 was restricted to the cytosol while form A4 occurred mainly in the mitochondrial matrix. Kidney arginases A1 and A4 were found to differ in immunological properties. Kidney arginase A1, in contrast to arginase A4, precipitated with antibodies against arginase A1 from rat liver. Arginase A1 from kidney was shown to differ from arginase A1 from the liver. The two enzymes could be distinguished by double diffusion test and immunoelectrophoresis.     

Expression of human liver arginase in Escherichia coli. Purification and properties of the product.

Arginase is an enzyme that catalyses the hydrolysis of arginine to urea and ornithine. It is abundantly present in the liver of ureotelic animals (i.e. those whose excretion is characterized by the excretion of uric acid as the chief end-product of nitrogen metabolism), but its purification has hitherto not been simple, and the yield not high. Starting with a partially truncated cDNA for human liver arginase recently made available, we constructed an expression plasmid that had tandemly linked tac promotors placed upstream of a full-length cDNA. By selecting Escherichia coli strain KY1436 as the host micro-organism, we established an efficient system for the production of human liver arginase protein. Chromatographies on CM-Sephadex G-150, DEAE-cellulose and Sephadex G-150, followed by preparative agar-gel electrophoresis, yielded 10 mg of apparently homogeneous enzyme protein from 1 g (wet wt.) of E. coli cells. E. coli-expressed human liver arginase had chemical, immunological and most catalytic properties indistinguishable from those of purified human erythrocyte arginase. However, E. coli-expressed arginase was a monomer of Mr 35,000, whereas the purified erythrocyte arginase was trimer of Mr 105,000. They differed also in pH- and temperature-stabilities. Gel-filtration experiments with these two purified arginases under various conditions, as well as with unfractionated human liver and erythrocyte cytosol preparations, indicated that the native form of human arginase should be of Mr 35,000, and that the trimeric appearance of human erythrocyte arginase after purification was an artifact of the purification procedures. It was thus concluded that, in Nature, the liver and erythrocyte arginases are identical proteins.     

Substrate inhibition of rat liver and kidney arginase with fluoride

Fluoride is an uncompetitive inhibitor of rat liver arginase. This study has shown that fluoride caused substrate inhibition of rat liver arginase at substrate concentrations above 4 mM. Rat kidney arginase was more sensitive to inhibition by fluoride than liver arginase. For both liver and kidney arginase preincubation with fluoride had no effect on the inhibition. When assayed with various concentrations of L-arginine, rat kidney arginase did not have Michaelis-Menten kinetics. Lineweaver-Burk and Eadie-Hofstee plots were nonlinear. Kidney arginase showed strong substrate activation at concentrations of L-arginine above 4 mM. Within narrow concentrations of L-arginine, the inhibition of kidney arginase by fluoride was uncompetitive. Fluoride caused substrate inhibition of kidney arginase at L-arginine concentrations above 1 mM. The presence of fluoride prevented the substrate activation of rat kidney arginase.     

Types of arginase in rat tissues.

Significant amounts of arginase activity were found in homogenates of submaxillary salivary gland and epididymis, as well as of liver, kidney, mammary gland, and small intestine. The isoelectric point of arginase solubilized from kidney was at pH 7.0 in contrast to that of pH 9.4 characteristic of hepatic arginase in rat. The isozymic variants of arginase in the different tissues were identified by their electrophoretic migration on polyacrylamide gels and by titration of the enzymes against antibody prepared against purified rat liver arginase. Antibody titrations confirmed the indications obtained by electrophoresis that one type of arginase is limited to hepatic tissues (and possibly submaxillary gland) while the other type is found in all other tissues. The physiological role of arginase in hepatic tissues has been previously associated with the urea cycle; the possible function of arginase in proline synthesis in other tissues remains to substantiated.     

Isolation of human liver arginase cDNA and demonstration of nonhomology between the two human arginase genes

A human liver cDNA library was screened by colony hybridization with a rat liver arginase cDNA. The number of positive clones detected was in agreement with the estimated abundance of arginase message in liver, and the identities of several of these clones were verified by hybrid-select translation, immunoprecipitation, and competition by purified arginase. The largest of these human liver arginase cDNAs was then used to detect arginase message on northern blots at levels consistent with the activities of liver arginase in the tissues and cells studied. The absence of a hybridization signal with mRNA from a cell line expressing only human kidney arginase demonstrated the lack of homology between the two human arginase genes and indicated considerable evolutionary divergence between these two loci.     

Cloning and expression in Escherichia coli of cDNA for arginase of rat liver

A cDNA expression library constructed in a plasmid pUC8 from poly(A)+ RNA of rat liver was screened immunologically, using an antibody against arginase of rat liver. A cDNA clone was isolated and identified by hybrid-selected translation. The clone contained an insert approximately 1.35 kilobase pairs in length. In the bacterial clone, we detected a specific protein of Mr = about 43,000 that is slightly larger than the purified arginase (Mr = about 40,000) and a high activity of arginase was expressed. The arginase mRNA species of about 1600 bases long was detected in the liver, but not in the small intestine, kidney, spleen and heart of the rats.     

Arginase expression and modulation of IL-1beta-induced nitric oxide generation in rat and human islets of Langerhans.

Proinflammatory cytokine induction of NO synthesis may contribute to the destruction of pancreatic beta cells leading to type 1 diabetes. The NO synthase substrate arginine can also be metabolized to ornithine and urea in a reaction catalyzed by cytosolic (AI) or mitochondrial (AII) isoforms of arginase. Recent evidence suggests that the rate of NO generation is dependent on the relative activities of NO synthase and arginase. The objectives of this study were (i). to identify the arginase isoforms expressed in rat and human islets of Langerhans and a rat beta cell line, RINm5F and (ii). to investigate the competition for arginine between NO synthase and arginase in IL-1beta-treated rat islets. Arginase activity was detected in rat islets (fresh tissue, 346 mU/mg protein; cultured, 587 mU/mg), cultured human islets (56 mU/mg), RINm5F cells (376 mU/mg), rat kidney (238 mU/mg), and rat liver (6119 mU/mg). Using Western blots, AI was shown to be the predominant isoform expressed in rat islets and in RINm5F cells while human islets expressed far more AII than AI. Rat islets were cultured in medium containing 1.14, 0.1, and 0.01 mM arginine and treated with IL-1beta and the arginase inhibitor 2(S)-amino-6-boronohexanoic acid (ABH). IL-1beta-induced NO generation was unaffected by ABH at 1.14 mM arginine, but significantly increased at 0.1 and 0.01 mM arginine. These findings suggest that the level of islet arginase activity can regulate the rate of induced NO generation and this may be relevant to the insulitis process leading to beta cell destruction in type 1 diabetes.     

Arginase expression and modulation of IL-1β-induced nitric oxide generation in rat and human islets of Langerhans

Proinflammatory cytokine induction of NO synthesis may contribute to the destruction of pancreatic beta cells leading to type 1 diabetes. The NO synthase substrate arginine can also be metabolized to ornithine and urea in a reaction catalyzed by cytosolic (AI) or mitochondrial (AII) isoforms of arginase. Recent evidence suggests that the rate of NO generation is dependent on the relative activities of NO synthase and arginase. The objectives of this study were (i) to identify the arginase isoforms expressed in rat and human islets of Langerhans and a rat beta cell line, RINm5F and (ii) to investigate the competition for arginine between NO synthase and arginase in IL-1β-treated rat islets. Arginase activity was detected in rat islets (fresh tissue, 346 mU/mg protein; cultured, 587 mU/mg), cultured human islets (56 mU/mg), RINm5F cells (376 mU/mg), rat kidney (238 mU/mg), and rat liver (6119 mU/mg). Using Western blots, AI was shown to be the predominant isoform expressed in rat islets and in RINm5F cells while human islets expressed far more AII than AI. Rat islets were cultured in medium containing 1.14, 0.1, and 0.01 mM arginine and treated with IL-1β and the arginase inhibitor 2(S)-amino-6-boronohexanoic acid (ABH). IL-1β-induced NO generation was unaffected by ABH at 1.14 mM arginine, but significantly increased at 0.1 and 0.01 mM arginine. These findings suggest that the level of islet arginase activity can regulate the rate of induced NO generation and this may be relevant to the insulitis process leading to beta cell destruction in type 1 diabetes.     

Inhibition of rat liver and kidney arginase by cadmium ion

Cadmium ion activates arginase from many species of organisms but is an inhibitor of arginase from many other species. The purpose of this study was to investigate the inhibition of rat liver and kidney arginase by cadmium ion. Rat kidney arginase was inhibited by much lower concentrations of cadmium ion than rat liver arginase. Cadmium ion was a mixed noncompetitive inhibitor of both rat liver and kidney arginase. Cadmium ion enhanced the substrate activation of rat kidney arginase while still inhibiting the enzyme. Cadmium ion prevented the substrate inhibition of rat kidney arginase by fluoride while still inhibiting the enzyme. Cadmium ion also inhibited rat kidney arginase in the presence of manganese ion.     

Inhibition of rat liver and kidney arginase by cadmium ion

Cadmium ion activates arginase from many species of organisms but is an inhibitor of arginase from many other species. The purpose of this study was to investigate the inhibition of rat liver and kidney arginase by cadmium ion. Rat kidney arginase was inhibited by much lower concentrations of cadmium ion than rat liver arginase. Cadmium ion was a mixed noncompetitive inhibitor of both rat liver and kidney arginase. Cadmium ion enhanced the substrate activation of rat kidney arginase while still inhibiting the enzyme. Cadmium ion prevented the substrate inhibition of rat kidney arginase by fluoride while still inhibiting the enzyme. Cadmium ion also inhibited rat kidney arginase in the presence of manganese ion.     

Functional heterogeneity among peritoneal macrophages: III. No evidence for the role of arginase in the inhibition of tumor cell growth by supernatants from macrophages or macrophage subpopulation cultures

The adherent population of peritoneal exudate cells (PE) obtained from rats and mice was analyzed for arginase activity in order to determine whether this enzyme has a role in tumor-growth-inhibitory activity. Freshly obtained tumor-growth-inhibitory rat PE cells had little or no arginase activity compared to the high levels of enzyme activity of mouse PE cells. Even after culturing, rat PE cells contained arginase activity 10 times less than that observed with comparable numbers of cultured or noncultured mouse cells. Subpopulations of mouse and rat PE macrophages, analyzed for arginase activity, showed that the light-density populations from cultured rat PE cells and noncultured mouse PE cells expressed arginase activities greater than that seen with heavy-density cells. However, the light-density rat PE cells expressed significantly less arginase activity than did the mouse cells. In attempts to test whether the inability of tumor cells to grow in supernatants or dialyzed supernatants from PE macrophage cultures is due to an arginine depletion, 200 μg/ml of the amino acid was added to the supernatants. The tumor-growth-inhibitory activities of such supernatants, as well as those from supernatants from highly active light-density rat PE macrophage cultures, were not abrogated by the addition of arginine. There was no correlation between the high levels of arginase activity of light-density PE macrophages and their antitumor activity and no evidence that the tumor-growth-inhibitory activity of rat or mouse PE macrophages in the macrophage-tumor models we studied was due to an arginine depletion.     

Production and characterization of monoclonal antibodies to rat liver arginase

Rat liver arginase was purified and five monoclonal antibodies were produced by fusion of spleen cells from a Balb/c mouse and the myeloma cell line P3-X36-Ag-U1. One, R2D19, of five antibodies belonged to the IgG2a subclass, the other four, R1D81, R1G11, R2E10, and R2G51, were of the IgG1 type. The R1D81 cross-reacted with human liver arginase. This antibody inhibited the arginase activity, competing with arginine. These results suggest that R1D81 binds to the catalytic site of arginase. The R2D19 also inhibited the enzyme activity but acted as a noncompetitive inhibitor. With the use of R1D81 and a polyclonal anti-human liver arginase antibody conjugated with alkaline phosphatase, a sandwich enzyme-linked immunosorbent assay (ELISA) was developed for the quantification of human arginase. Specificity of monoclonal antibodies for rat liver arginase was examined by means of the sandwich ELISA. Eight pairs of monoclonal antibodies could form a sandwich with the arginase. Only the R2E10 could be used for both the first and the second antibody in the sandwich system. In other cases, monoclonal antibodies could not be interchanged between solid and liquid phase.     

Studies on the advent of ureotelism. Factors that render the hepatic arginase of the Mexican axolotl able to hydrolyse endogenous arginine

1. A study was undertaken of the conditions that might operate in the synthesis and hydrolysis of arginine by axolotl liver homogenate to test a previous postulate that liver arginase of the non-metamorphosed Mexican axolotl is not able to hydrolyse arginine formed from citrulline and aspartic acid, though it can split exogenous arginine, and also that an enhanced capacity to hydrolyse endogenous arginine plays a major role in the advent of ureotelism observed during the metamorphosis of the axolotl. 2. It was found that the arginase from axolotl liver is very unstable under the conditions followed, contrary to what is observed in rat liver. 3. Axolotl arginase is able to hydrolyse endogenous arginine if preserved. 4. Mn(2+) protects the enzyme and renders it able to split endogenous arginine. 5. It is suggested that the metal ion produces a change of conformation of the enzyme that, being stable, is capable of hydrolysing the amino acid, or that the new conformation is appropriate for interaction with the sites of arginine synthesis.     

Identification of the apparently lymphocyte-specific human liver-derived inhibitory protein (LIP) as cytoplasmic liver L-arginase

This paper describes the identification of a human liver-derived inhibitory protein (LIP), which has recently been purified, as cytoplasmic liver arginase. Arginase activity was purified to homogeneity parallel to lymphocyte proliferation inhibitory activity. The reaction products were identified by thin-layer chromatography to be ornithine and urea from arginine. The enzyme activity could be increased by the addition of manganese ions, and the inhibitory effect on cell proliferation could be reversed by additional arginine. An antiserum against LIP cross-reacted with cytoplasmic calf liver arginase.     

Tissue-specific differential modulation of arginase and ornithine aminotransferase by hydrocortisone during various developmental stages of the rat

The activities and regulatory patterns of arginase and ornithine aminotransferase (OAT) of the liver (a mitotic tissue) and kidney cortex (a post-mitotic tissue) of immature, adult, and senescent male rats were studied. The activities of the liver enzymes were highest in the immature rat and decreased gradually with age. However, in the kidney cortex, the activity of arginase was highest and decreased significantly thereafter while that of OAT shows no significant change throughout the life span of the rat. Further, the activity of kidney cortex arginase was approximately 1/20th of that of the liver enzyme. Adrenalectomy and hydrocortisone treatments altered the activity of arginase in both tissues and that of OAT in the liver only. However, the kidney cortex OAT was not responsive towards these treatments. Actinomycin D inhibited the hydrocortisone-mediated induction of arginase of both the liver and kidney cortex and that of the liver OAT.