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.     

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 and characterization of alkaline phosphatase from rat kidney.

Alkaline phosphatase [EC 3.1.3.1.] was purified about 250-fold from rat kidney, and its enzymological properties were studied. Kidney homogenate was extracted with n-butanol, passed through Sephadex G-200 and chromatographed on a DEAE-cellulose column. The peak from the DEAE-cellulose column was subjected to isoelectric focusing, and the alkaline phosphatase activity was separated into two peaks. The molecular weights of alkaline phosphatase in these peaks were 4.8.X10(4) and 1.0X10(5), as determined by SDS-polyacrylamide gel electrophoresis. Anti-serum against alkaline phosphatase from rat kidney was prepared, and was shown to neutralize the activity from kidney, liver or bone, but not that from intestine.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Rat Mammary Arginase: Isolation and Characterization

The extrahepatic arginase, AII, from rat mammary gland was isolated and its properties investigated and compared with those of the hepatic arginase, AI. Mammary arginase activity increased 300% at mid-lactation, an increase unaccompanied by an increase in liver arginase activity. Mammary gland contained two isozymes, separable by ion exchange chromatography. The major form, AII, was purified 103-fold and antisera were raised against it. A 1300-fold purification was achieved temporarily but the enzyme was unstable. Arginase AII was kinetically similar to AI: both had pH optima of 10 and K_ms for L-arginine of 12-14 mM. Arginase AII differed from AI in having a near-neutral pI and a slightly larger subunit size (39,800 Da compared to 38,900 Da by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)). Solution immunoprecipitation studies revealed that virtually all of the arginase present in liver was type AI, whereas kidney and mammary gland contained both isozymes. Western immunoblotting showed that the amount of immunoreactive mammary arginase AII protein increased at mid-lactation in parallel with the increase in activity. This suggests that the elevated arginase activity is due to de novo protein synthesis and/or reduced protein degradation, rather than activation of arginase.     

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.     

以酶标免疫吸附法测定大鼠肝脏精氨酸酶含量

本文利用丙酮沉淀、凝胶过滤、离子交换层析等方法提纯了大鼠肝脏精氨酸酶,在SDS-PAGE中表现为单一的蛋白质带,其亚基分子量为37,000.建立了大鼠肝精氨酸酶的酶标免疫吸附测定法(ELISA),比较了两种常规的精氨酸酶活性测定法与ELISA法,并初步探讨了ELISA法在精氨酸酶测定方面的一些应用。     

The relation of the soluble thiamine triphosphatase activity of various rat tissues to nonspecific phosphatases

Polyacrylamide gel electrophoresis was used to investigate the relation of the soluble thiamine triphosphatase activity of various rat tissues to other phosphatases. This technique separated the thiamine triphosphatase of rat brain, heart, kidney, liver, lung, muscle and spleen from alkaline phosphatase (EC 3.1.3.1), acid phosphatase (EC 3.1.3.2) and other nonspecific phosphatase activities. In contrast, the hydrolytic activity for thiamine triphosphate in rat intestine moved identically with alkaline phosphatase in gel electrophoresis. Thiamine triphosphatase from rat liver and brain was also separated from alkaline phosphatase and acid phosphatase by gel chromatography on Sephadex G-100. This gave an apparent molecular weight of about 30,000 and a Stokes radius of 2.5 nanometers for brain and liver thiamine triphosphatase. The intestinal thiamine triphosphatase activity of the rat was eluted from the Sephadex G-100 column as two separate peaks (with apparent molecular weights of over 200,000 and 123,000) which exactly corresponded to the peaks of alkaline phosphatase. The isoelectric point (pI) of the brain thiamine triphosphatase was 4.6 (4 degrees C). The partially purified thiamine triphosphatase from brain and liver was highly specific for thiamine triphosphate. The results suggest that, apart from the intestine, the rat tissues studied contain a specific enzyme, thiamine triphosphatase (EC 3.6.1.28). The specific enzyme is responsible for most of the thiamine triphosphatase activity in these tissues. Rat intestine contains a high thiamine triphosphatase activity but all of it appears to be due to alkaline phosphatase.     

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.     

Soluble metalloendopeptidase (MMP-7ase) activity in mouse kidney cytosol.

Previously, MMP-7ases were isolated from rat skeletal muscle by gel filtration and anion exchange chromatography. The enzyme that hydrolyzed succinyl-Ala-Ala-Pro-Phe-AMC (AMC: 7-amino-4-methyl-coumarin) was inhibited by EDTA. In this study we attempted to isolate MMP-7ase from mouse kidney. The isolation procedure was the same as that previously used for skeletal muscle. Kidneys of ICR mice were homogenized and, after centrifugation, the supernatant fraction was subjected to gel filtration chromatography. The fraction with the highest activity (Mr 67-72 kDa) was subjected to anion exchange chromatography, which showed three peaks of activity. The second peak hydrolyzed succ-Ala-Ala-Pro-Phe-AMC, but had low activity against Arg- or Ala-AMC. This peak was a single protein (Mr 68-72 kDa) and its activity could be inhibited with EDTA. Several tri- and tetrapeptide derivatives were tested as substrates for this enzyme and the best was found to be succ-Ala-Ala-Pro-Phe-AMC. We can conclude that mouse kidney cytosol contains a metalloendopeptidase similar to muscle MMP-7ase.     

Resolution of multiple forms of bovine liver arginase by chromatofocusing

1. Bovine liver arginase could be resolved into three distinct peaks by chromatofocusing in the pH range 7-4. 2. In other experimental systems the enzyme appeared to consist of a single active component. 3. Sodium dodecylsulphate-polyacrylamide gel electrophoresis revealed a single band which could be assigned to arginase, with no indication of inherent or protease-induced multiplicity. 4. Lineweaver-Burk plots for arginine were linear over a wide concentration range, as were Dixon plots for reversible inhibitors. 5. Covalent inhibition by 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide gave semilogarithmic plots of residual activity vs time which were strictly linear. 6. It was concluded that the enzyme was homogeneous with respect to subunit size and kinetic behaviour, but heterogeneous with respect to molecular charge. 7. The charge heterogeneity may have kinetic and regulatory implications, as previously suggested for mouse liver arginase [Z. Spolarics and J. S. Bond (1988) Archs Biochem. Biophys. 260, 469-479].     

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.     

Induction of rat hepatic alkaline phosphatase and its appearance in serum: electrophoretic characterization of liver-membranous and serum-soluble forms

Simultaneous bile duct ligation and colchicine injection (2 mg/kg body weight) in rats caused a remarkable induction of alkaline phosphatase in the liver. Concomitantly, a marked elevation of the enzyme activity occurred in the serum, and three activity peaks (peaks I, II, and III) were separated by Sephadex G-200 gel filtration. By several criteria for alkaline phosphatase isoenzymes it was determined that the liver-derived enzyme was distributed in peak I (30% of total serum activity) as a vesicle-bound form and in peak II (65%) as a soluble form, while the intestinal enzyme was contained in peak III (5%). The serum alkaline phosphatase in peaks I and II was compared with the liver enzyme extracted from plasma membrane with n-butanol. Under non-reducing conditions, the soluble form of peak II showed an electrophoretic mobility different from that of the liver enzyme; in the presence of sodium dodecyl sulfate the serum-soluble form migrated a little more slowly than the liver one, while in the presence of Triton X-100 the former migrated much faster than the latter. The sedimentable fraction of peak I was found to contain two forms corresponding to the serum-soluble and liver-membranous forms. Neuraminidase treatment of these two forms reduced their mobilities but did not abolish the relative difference in their mobilities on gel electrophoresis in the presence of either Triton X-100 or sodium dodecyl sulfate. Under reducing conditions, however, each form (which was dissociated into single subunits) migrated with an identical mobility on sodium dodecyl sulfate gel electrophoresis. These results suggest that the hepatic alkaline phosphatase exists as conformationally different forms in the serum and the liver membrane (even solubilized), but the difference is no longer preserved after their denaturation into subunits.