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.     

Five forms of arginase in human tissues

Five forms of arginase, A1, A2, A3, A4, and A5, were found to be present in human tissues. The molecular weight of all these forms is the same, 120,000, but they differ in the behavior on DEAE- and CM-cellulose, electrophoretic mobility, isoelectric point, and immunochemical properties. Forms A1 from kidney and A5 from liver show complete immunological incompatibility, whereas forms A2 from liver, A3 from salivary gland, and A4 from kidney exhibit partial incompatibility with respect to each other and to forms A1 and A5.     

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.     

Hybridization of subunits of rat liver arginase A1 and rat kidney arginase A4

Recombination of subunits of rat liver arginase A1 and rat kidney arginase A4 yielded a product which in polyacrylamide gel electrophoresis and DEAE-cellulose chromatography separated into five proteins with arginase activity. Proteins I and V corresponded in polyacrylamide gel-electrophoresis, DEAE-cellulose chromatography and immunological properties to the parental forms A1 and A4, respectively. Formation of five arginase hybrids proved the tetrameric structure of native arginases.     

Immunological properties of rat arginases

Five immunologically different forms of arginase were evidenced in rat tissues by the double diffusion test and immunoelectrophoresis. New symbols for these arginases are proposed (beginning with the most anionic forms): A1 (kidney), A2 (liver), A3 (salivary glands), A4 (kidney) and A5 (liver). Arginases A1 from kidney and A5 from liver are paternal forms built of one-type subunits. Subunits of form A1 exhibit a non-identity cross-reaction with subunits of form A5. Arginases A2, A3 and A4 are hybrids composed of both kinds of subunits.     

Differential expression of multiple forms of arginase in cultured cells

Summary Arginase (EC 3.5.3.1), the final enzyme in the urea cycle, catalyzes the cleavage of arginine to orthinine and urea. At least two forms of this enzyme, Al and All, have been described and are probably encoded by discrete genetic loci. The expression of these separate genes has been studied in mammalian cells grown in culture. The permanent rat-hepatoma line H4-II-E-C3 contained exclusively the Al enzyme; the form in mammals comprising about 98% of the arginase activity in liver and erythrocytes but catalyzing only about one half of that reaction in kidney, gastrointestinal tract, and brain. By contrast, human-embryonic-kidney and -brain cells, after transformation with the human papovavirus BK, contained only the All species of arginase, which form contributes the remaining half of that catalysis in those mammalian tissues in vivo. We report here the results of an extensive study on the properties of these two forms of arginase in the three cell lines, including K_m values for arginine, behavior on polyacrylamide gels under non-denaturing conditions, and cross-reactivity with lapine antibodies against the arginases from either rat or human liver.[/p]Presented in part at the annual meeting of the Society for Pediatric Research, Washington, D.C., May, 1982. Pediatr. Res. 16:195A.     

Immunohistochemical localisation of arginase in human liver using monoclonal antibodies against human liver arginase

Monoclonal antibodies against human liver arginase were raised in order to determine the exact distribution of arginase in human liver using a modified indirect unlabelled immunoperoxidase method. In normal human liver specific immunohistochemical staining was found in the cytoplasm of hepatocytes. Portal components (bile ducts and veins) and fibrous tissue were non-reactive, while erythrocytes were slightly positive. The specificity of the immunological reaction was confirmed by control tests. Spectrophotometry was used to quantitate the immunohistochemical reaction product, and the results indicated that arginase is homogeneously distributed in the liver lobule.     

Comparison of biochemical properties of liver arginase from streptozocin-induced diabetic and control mice

Arginase activity is elevated in livers of diabetic animals compared to controls and there is evidence that this is due in part to increased specific activity (activity/mg arginase protein). To investigate the molecular basis of this increased activity, the physicochemical and kinetic properties of hepatic arginase from diabetic and control mice were compared. Two types of arginase subunits with molecular weights of 35,000 and 38,000 were found in both the diabetic and control animals and the subunits in these animals had similar, multiple ionic forms. Kinetic parameters of purified preparations of arginase for arginine (apparent Km and Vmax values) and the thermal stability of these preparations from diabetics and controls were also similar. Furthermore, no difference was found in the distribution of arginase activity among different subcellular liver fractions. Separation of basic and acidic oligomeric forms of arginase by fast-protein liquid chromatography resulted in a slightly different distribution of activity among the forms in the normal and diabetic group. The apparent Km values for Mn2+ of the basic form of the enzyme were 25 and 33 microM for the enzyme from normal and diabetic animals, respectively; for acidic forms, for which two apparent Km values were measured, the values were 8 and 197 microM for arginase from controls and 35 and 537 microM from diabetics. These results indicate that in diabetes, while no marked changes in the physicochemical characteristics of arginase are obvious, some changes are found in the interaction of arginase with its cofactor Mn.     

Purification and properties of arginase of rat kidney

l-Arginase from rat kidney was partially purified and some properties were compared with those of l-arginase of rat liver. The kidney enzyme was firmly bound to the mitochondrial fraction and after solubilization required arginine or an unknown factor in tissue extracts for stabilization after dialysis. The two enzymes differed also in stability with respect to acetone treatment, heating or freezing. In further contrast with liver arginase, arginase from kidney was not adsorbed to CM-cellulose at pH7.5 and its activity was not increased by incubation with Mn(2+). Other differences were seen in relative specificities for substrates, ratio of hydrolysis rates with high and low concentrations of arginine and effects of certain inhibitors. Antisera prepared to pure liver arginase did not cross-react with partially purified kidney arginase.     

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.     

Immunohistochemical localisation of arginase in human liver using monoclonal antibodies against human liver arginase

Summary Monoclonal antibodies against human liver arginase were raised in order to determine the exact distribution of arginase in human liver using a modified indirect unlabelled immunoperoxidase method. In normal human liver specific immunohistochemical staining was found in the cytoplasm of hepatocytes. Portal components (bile ducts and veins) and fibrous tissue were non-reactive, while erythrocytes were slightly positive. The specificity of the immunological reaction was confirmed by control tests. Spectrophotometry was used to quantitate the immunohistochemical reaction product, and the results indicated that arginase is homogeneously distributed in the liver lobule.Present address: Biologisches Institut der Universität Stuttgart, Ulmerstrasse 227, D-7000 Stuttgart 60, Federal Republic of Germany     

Arginase isoforms in frog and lizard tissues

Isoforms of arginase in the liver and kidney tissues of the ureotelic frog (Rana tigerina) and uricotelic lizard (Calotes versicolor) were fractionated by DEAE-cellulose chromatography (pH 8.3). Four molecular forms, designated as A'1, A2, A3 and A4 based on the KCl concentration required for their elution from the ion-exchange column, were detected in lizard liver, while only two forms were found in lizard kidney (A3 and A4) and frog liver and kidney (A2 and A3). No major differences were found in the pH optimum, substrate affinity and molecular weight of the isoenzymes. The isoforms in lizard tissues were either totally unaffected or only partially immunoprecipitated by antibodies raised against rat liver and beef liver arginases, but those in frog tissues were significantly activated by the two antibodies. While the physiological importance of the presence of four isoforms in lizard liver remains enigmatic, different sets of isoenzymes were present in the liver of the two ureotelic vertebrates, rat and frog. Hence, it appeared that a given mode of nitrotelism was not associated with a specific set of isoenzymes. Also, the data were not consistent with the generally held view that a basic isoform of arginase served as a component of the urea cycle in liver and a neutral/slightly acidic form functions in the synthesis of proline, glutamate and polyamines in extra-hepatic tissues. The isoforms appeared to show considerable functional overlap.     

Studies on L-arginase in developing rat small intestine, brain, and kidney. I. Ontogenic evolution of arginase isoenzymes

The adult patterns of arginase isoenzymes in rat intestine, kidney, and brain are nearly identical and consist of two forms, cationic A1 and anionic A4. In this paper, the organ-specific maturation of the enzyme equipment in these tissues is reported. The activity of arginase in all tissues studied could be detected on the 13th to 16th days of gestation. In fetal intestine and kidney the arginase activity is low, and persists up to the weaning time when the rapid, 10-fold rise of the enzyme activity occurs. However, the adult pattern of arginase isoenzymes in these tissues is accomplished in different ways. In the intestine, arginase A1 appears in fetal life and is the only form of the enzyme till the 19th to 21st days of postnatal life when the second form of arginase, A4, appears and rapidly accumulates, being exclusively responsible for the rise of the total enzyme activity at the time of weaning. In kidney, arginase A1 alone is present in the early fetal period. Arginase A4 appears 3-4 days before birth and its activity persists unchanged within the first 2 weeks of postnatal life. The intensive rise in total specific activity of kidney arginase at weaning is due to the accumulation of preexisting arginase A4. In brain, the adult pattern of arginase isoenzymes is achieved earlier than in other tissues. Both forms, A1 and A4, occur on Days 13-14 of gestation.     

Two distinct forms of glutathione transferase from human foetal liver. Purification and comparison with isoenzymes isolated from adult liver and placenta.

Isoelectric focusing of a cytosol fraction from human foetal liver revealed the existence of an acidic and a basic isoenzyme of GSH transferase. The acidic and basic forms of GSH transferase were purified in good yield by use of ion-exchange chromatography on DEAE-cellulose followed by affinity chromatography on S-hexyl-GSH coupled to epoxy-activated Sepharose 6B. The content of the acidic and the basic isoenzymes of GSH transferase together was calculated to constitute 1-2% of the soluble proteins in the hepatic cytoplasm. Physical, catalytic and immunological analyses of the acidic and the basic isoenzymes from foetal liver demonstrated unambiguously that the two forms are different structures with distinct properties. On the other hand, the results show clearly extensive similarities between the foetal acidic transferase and transferase pi from human placenta as well as between the foetal basic form and the basic isoenzymes isolated from adult liver. An exception is that both foetal enzymes seem to be considerably more efficient in catalysing the conjugation of GSH with styrene 7,8-epoxide than the corresponding adult forms of GSH transferase.     

Tissue cholinesterases. A comparative study of their kinetic properties

The substrate saturation and temperature-dependent kinetic properties of soluble and membrane-bound forms of acetylcholinestarase (AChE) from brain and butyrylcholinesterase (BChE) from heart and liver were examined. In simultaneous studies these parameters were also measured for AChE in erythrocyte membranes and for BChE in the serum from rat and humans. For both soluble and membrane-bound forms of the enzyme from the three tissues, two components were discernible. In the brain, Km of component I (high affinity) and component II (low affinity) was somewhat higher in membrane-bound form than that of the soluble form components, while the Vmax values were significantly higher by about five fold. In the heart, Km of component II was lower in membrane-bound form than in the soluble form, while Vmax for both the components was about four to six fold higher in the membrane-bound form. In the liver, Vmax was marginally higher for the two components of the membrane-bound enzyme; the Km only of component I was higher by a factor of 2. In the rat erythrocyte membranes three components of AChE were present showing increasing values of Km and Vmax. In contrast, in the human erythrocyte membranes only two components could be detected; the one corresponding to component II of rat erythrocyte membranes was absent. In the rat serum two components of BChE were present while the human serum was found to possess three components. Component I of the human serum was missing in the rat serum. Temperature kinetics studies revealed that the Arrhenius plots were biphasic for most of the systems except for human serum. Membrane binding of the enzyme resulted in decreased energy of activation with shift in phase transition temperature (Tt) to near physiological temperature.     

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.     

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.     

Arginase of Agrobacterium Ti plasmid C58. DNA sequence, properties, and comparison with eucaryotic enzymes

Agrobacterium nopaline Ti plasmids code for three enzymes of nopaline [N2-(1,3-dicarboxypropyl)-L-arginine] degradation: nopaline oxidase, arginase, and ornithine cyclodeaminase. We describe the DNA sequence of the arginase gene, a comparison of the deduced protein sequence with eucaryotic arginases, and properties of the procaryotic enzyme. The results show that the agrobacterial arginase is related with arginases from yeast, rat liver, and human liver (28-33% identity). The Ti plasmid enzyme revealed several properties which appear common to all arginases, but it does not utilize L-canavanine as substrate, and its Mn2+ requirement is not satisfied by Fe2+, Co2+, or Ni2+. The properties of arginase and ornithine cyclodeaminase are discussed as part of the mechanisms which avoid depletion of L-arginine and L-ornithine pools for biosynthetic reactions during catabolic utilization of nopaline.     

Pyruvate kinase isozymes in man

Summary By focusing in sucrose, gradient L-type pyruvate kinase from human liver could be separated into 2 major forms (pI 6.28±0.03 and 5.85±0.09) and a minor more acid form (pI5). These different forms could also be detected by focusing in acrylamide-ampholine slab gel. The major forms were interconvertible, the equilibrium being shifted toward the acid form by fructose 1,6-diphosphate and SH reagents, and toward the alkaline form by proteinic factors extracted by ammonium sulphate fractionation from liver extracts and from hemolysates. These factors seemed to be responsible for the stabilization of the liver crude extract enzyme in its alkaline conformation.By acrylamide slab gel electrofocusing, erythrocyte pyruvate kinase from whole hemolysates exhibited a complex pattern composed of at least 3 interconvertible forms. The in vitro aging of the red blood cells and the storage of the hemolysates resulted in a progressive disappearance of the acid forms and in a strengthening of the alkaline form. Partially purified erythrocyte enzyme focused in 2 major bands, interconvertible under the influence of the same factors as those described for L-type pyruvate kinase. Although closely related, the focusing patterns of L-type and erythrocyte-type were never exactly identical.Double immunodiffusion against antihuman L-type serum showed a complete identity reaction between erythrocyte-and L-type pyruvate kinases. Moreover, antihuman M₂-type serum was unable to neutralize erythrocyte pyruvate kinase as well as to change its electrophoretic mobility.Consequently, we conclude that both human erythrocyte-and liver L-type pyruvate kinases existed under several conformers interconvertible under the influence of the same ligands or proteinic factors; erythrocyte-type enzyme seems to include L-type subunit and not M₁- or M₂-type subunits. The erythrocyte- and L-type enzymes, however, are not identical and the nature of the differences between them is discussed.Chargé de recherche INSERM.