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.     

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.     

The isolation and immunological properties of two arginase forms from human erythrocytes

1. Two forms of arginase were isolated from human erythrocytes; the main form adsorbed on CM-cellulose and the second form, occurring in much smaller amount, adsorbed on DEAE-cellulose. 2. The molecular weight of either arginase was 120,000 +/- 5000. 3. The erythrocyte arginases are similar in immunological properties to arginase A4 from human kidney and A2 from human liver, respectively. 4. Despite the literature data stating that human erythrocyte arginase and human liver arginase are identical, it was found that the main forms of arginase of these tissues A4 from erythrocytes and A5 from liver differ in immunological properties.     

Multiple molecular forms of mouse liver arginase

Hepatic arginase (L-arginine amidinohydrolase, EC 3.5.3.1) is an oligomer composed of three or four subunits. The present studies indicate heterogeneity in the size and charge of arginase subunits in mouse liver. Two types of arginase subunits with molecular weights of approximately 35,000 and 38,000 have been found. These two subunits are detected in liver cytosol or in purified preparations of arginase after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. Two dimensional SDS-PAGE revealed multiple ionic forms of arginase for both the 35,000 and 38,000 subunits; the subunits contain basic proteins (pI range 7.8-9.1) and acidic proteins (pI range 5.8-6.4). Limited proteolysis by trypsin eliminated the molecular weight differences between the subunits without substantially affecting either their isoelectric points or activity. Comparative peptide maps and amino acid analyses of the 35,000- and 38,000-Da subunits showed that they were very similar. The data indicate that a neutral peptide (approx 3000 Da) is responsible for the differences in subunit molecular weight and that the multiple sized and charged forms are variants of the same protein.     

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.     

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     

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.     

Expression of three forms of thyroid hormone receptor in human tissues

At least two thyroid hormone receptor (hTR) genes are present in humans, but the significance of this multiplicity is unknown. These receptors could have differences in tissue distribution or possess different functions. We studied the distribution and abundance of three hTR mRNAs (hTR beta, hTR alpha 1, and hTR alpha 2) by Northern blot analysis. Three mRNAs were expressed in all tissues examined. hTR beta was strongly expressed in brain and prostate predominantly as a 10.0-kilobase (kb) mRNA. This mRNA was also expressed in thyroid and was much less abundant in liver, kidney, placenta, tonsil, and spleen. hTR alpha 1 is represented by two mRNAs with sizes of 6.0 and 3.2 kb. The 6.0-kb mRNA was constantly less abundant than the 3.2-kb mRNA. hTR alpha 2 was detected as a single mRNA with a size of 3.2 kb, using a probe unique for this mRNA. Both hTR alpha 1 and hTR alpha 2 were strongly expressed in brain, prostate, and thyroid and much less in other tissues. The relative amounts of the three hTR mRNAs were roughly parallel in each tissue. It is of interest that none of these hTRs was abundant in liver, which is the major thyroid hormone-responsive organ. Another hTR may be present in liver.     

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].     

Automated determination of arginase in human erythrocytes

An automated technique is reported for measuring erythrocyte arginase activity by direct reaction of urea and diacetylmonoxime. The effects of pH, temperature, substrate concentration, activator, interference, enzyme stability, and reproducibility are examined and discussed.     

A comparative polyacrylamide gel electrophoretic study of arginase in vertebrate tissues

The electrophoretic behaviour of arginase in the tissue extracts of rat, beef, lizard and frog was studied by bidirectional polyacrylamide gel electrophoresis. The enzyme from rat liver and submaxillary gland migrated to the cathode with the activity concentrated in a single peak. Arginase from beef liver emerged as a single peak of anodal migration with a significant shoulder in the sample gel. Frog liver and kidney enzymes also appeared as single peaks with a distinct anodal movement. The activity in mammalian kidney and lizard liver and kidney resolved into two peaks of anodal migration suggesting the presence of two isoenzymes of arginase in these tissues.     

Inhibition profile of Leishmania mexicana arginase reveals differences with human arginase I

Arginase (ARG), the enzyme that catalyzes the conversion of arginine to ornithine and urea, is the first and committed step in polyamine biosynthesis in Leishmania. The creation of a conditionally lethal Δarg null mutant in Leishmania mexicana has established that ARG is an essential enzyme for the promastigote form of the parasite and that the enzyme provides an important defense mechanism for parasite survival in the eukaryotic host. Furthermore, human ARGI (HsARGI) has also been implicated as a key factor in parasite proliferation. Thus, inhibitors of ARG offer a rational paradigm for drug design. To initiate a search for inhibitors of the L. mexicana ARG (LmARG), recombinant LmARG and HsARGI enzymes were purified from Escherichia coli. Both LmARG and HsARGI were specific for l-arginine and exhibited no activity with either d-arginine or agmatine as possible substrates. LmARG exhibited a K_m of 25 ± 4 mM for l-arginine, a pH optimum ∼9.0, and was dependent upon the presence of a divalent cation, preferentially manganese. A K_m of 13.5 ± 2 mM for l-arginine was calculated for the HsARGI. A collection of 37 compounds was evaluated against both enzymes. Twelve of these compounds were identified as being either strong inhibitors of both LmARG and HsARGI or differential inhibitors between the two enzymes. Of the 12 compounds, six were selected for further analysis and the type and extent of inhibition determined.