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.     

The resolution and characterization of putative ubiquitin carrier protein isozymes from rabbit reticulocytes

The covalent ligation of the 8.6-kDa polypeptide ubiquitin to various cellular target proteins is believed to represent a fundamental regulatory process. In this mechanism, the ATP-coupled activation and subsequent ligation of ubiquitin are catalyzed by separate enzymes (E1 and E3, respectively) functionally linked by ubiquitin carrier protein (E2). Carrier protein has been proposed to constitute a family of isozymes having molecular masses of 14, 17, 20, 24, and 32 kDa whose role is to shuttle activated polypeptide in the form of a high-energy thiol ester intermediate to the carboxyl terminus of ubiquitin. Using a combination of covalent affinity and high performance liquid chromatographic methods, the pututive E2 isozymes have been purified to apparent homogeneity. The E2(14kDa) isozyme resolved into two forms differing in net charge at pH 7.5. All of the E2 isozymes contained only one thiol ester site except for E2(17kDa) and E2(20kDa) which were capable of forming two such adducts per subunit. Thiol ester formation was rapid for the E2 isozymes and required the presence of activating enzyme. In contrast, the reverse reaction of thiol ester transfer from E2 to E1 was kinetically significant for only E2(14kDa), E2(20kDa), and E2(24kDa). The stability of E2(17kDa) and E2(32kDa) to such trapping may reflect a marked shift in binding affinity to E1 upon thiol ester formation. In addition, differential rates for thiol ester formation to each subunit of dimeric E2(14kDa) was also noted. The E2(14kDa) isoforms were approximately 10-fold more active in E3-dependent ubiquitin-protein ligation than either E2(20kDa) or E2(32kDa). Neither E2(17kDa) nor E2(24kDa) supported this reaction. In addition, the thiol ester formed to E2(14kDa) was inherently more reactive since its second order rate constant for the E3-independent transfer of ubiquitin to the small molecular weight nucleophile dithiothreitol was an order of magnitude greater than found for the other isozymes. If these proteins constitute a family of isozymes, they exhibit considerable catalytic diversity.     

Human hyperargininemia: a mutation not expressed in skin fibroblasts?

Arginase specific activity in the fibroblasts from three hyperargininemia patients is similar to that in controls. Kinetic features, pH-optimum, effect of Mn++, apparent Km values and DEAE- and CM-cellulose chromatography isozymes are identical in either cell type. The arginase gene functional in fibroblasts may be unrelated to the cause of hyperargininemia in humans. The latter mutation may solely affect the arginase of erythrocytes.     

The mitochondrial connection: Arginine degradation versus arginine conversion to nitric oxide

Arg catabolism to cytoplasmic urea and glutamate is initiated by two mitochondrial enzymes, arginase and ornithine aminotransferase. Mutation of either enzyme leads to Arg sensitivity, and at least in the former, an arginine-induced seedling morphology similar to exogenous auxin treatment. We reported that single mutants lacking either of two arginase isozymes exhibited more NO accumulation and efflux, and increased responses to auxin (measured by DR5 reporter expression and auxin-induced lateral roots). We discuss evidence for stimulation of NO by arginine, either directly, or via polyamines derived from arginine. We favor the “direct” route because mitochondria are sites of NO ‘hot spots,’ and the location of arginine-degrading enzymes and the NO-associated protein1. The polyamine “branch” invokes more than one cell compartment, at least two intermediates (polyamines and H₂O₂) between Arg and NO, and is not consistent with enhanced lateral root formation in arginine decarboxylase mutants. Genetic tools are at our disposal to test the two possible routes of arginine-derived NO.Key words: nitric oxide, arginine, arginase, root development, polyamines, auxins     

Design,synthesis and biological evaluation of trinary benzocoumarin-thiazoles-azomethines derivatives as effective and selective inhibitors of alkaline phosphatase

Design, synthesis and characterization of new trinary Benzocoumarin-Thiazoles-Azomethine derivatives having three bioactive scaffolds in a single structural unit were carried out. The newly synthesized molecules were investigated for the inhibitory activity on human tissue nonspecific alkaline phosphatase (h-TNAP) and human intestinal alkaline phosphatase (h-IAP) isozymes. All the tested compounds exhibited the potent inhibition profile on both isozymes of alkaline phosphatase i.e., h-TNAP and h-IAP. Molecular docking studies were performed to explore the putative binding mode of interactions of selective inhibitors. Moreover, the synthesized derivatives were evaluated against cervical cancer cell line, HeLa and a few compounds exhibited significant inhibition in the range of 21.0–69.7%. The derivatives can be potential and selective alkaline phosphatase inhibitors for future studies.     

Preparative separation of hemoglobins A and S by gel electrofocusing, using selective zone elution by gel transposition between suitable anolytes and catholytes.

Preparative electrofocusing on polyacrylamide gels has been limited, until recently, to excision of gel slices, diffusion, and collection of the slice diffusates. An advance was made by the introduction of a method of selective electrophoretic zone recovery by specific changes of anolyte (A. McCormick, L. E. M. Miles, and A. Chrambach, 1976, Anal. Biochem.75, 314–324). It was shown (a) that selective zone recovery could be achieved by transposition of the gels into either isoelectric ampholytes or charged buffers, (b) that it could be applied to the gram scale, and (c) that zone elution could proceed either continuously or discontinuously. The early study was, however, limited to a trivial model problem, the separation of hemoglobin from bovine serum albumin (BSA). The present study was an attempt to apply a similar selective zone recovery method to a more demanding separation problem, the separation of hemoglobin A from hemoglobin S as well as from other minor components contained in a sickle-trait human hemolysate. The study shows that selective electrophoretic zone elution from a electrofocusing gel 18 mm in diameter is capable of yielding hemoglobin A, separated from hemoglobin S, differing by only 0.2 pH units in isoelectric point. The recovery of hemoglobin A was 70%, with a load of 32 mg of hemoglobin mixture per gel, using discontinuous zone elution into a collection cup.     

Biochemical and molecular characterization of mitochondrial membrane-bound arginase in <Emphasis Type="Italic">Heteropneustes fossilis</Emphasis>

The two predominant forms of arginase, cytosolic Arginase-I and mitochondrial Arginase-II, catalyze hydrolysis of arginine into ornithine and urea. Based on presence of arginase activity in extracts using potassium chloride (KCl), mitochondrial membrane-bound arginase has also been suggested. However, the activity of arginase in fractions obtained after KCl-treatment may be either due to leakage of mitochondrial arginase or release of adhered cytosolic arginase to cell organelles having altered net charge. Therefore, it has been intended to analyse impact of KCl on ultra-structural properties of mitochondria, and biochemical analysis of mitochondrial membrane-bound proteins and arginase of Heteropneustes fossilis. Liver of H. fossilis was used for isolating mitochondria for analysis of ultrastructural properties, preparing cytosolic, mitochondrial, and mitochondrial-membrane bound extracts after treatment of KCl. Extracts were analysed for arginase activity assay, protein profiling through SDS-PAGE and MALDI MS/MS. The KCl-mediated modulation in polypeptides and arginase were also evaluated by PANTHER, MitoProt and IPSORT servers. The effects of KCl on ultra-structural integrity of mitochondria, activity of arginase, modulation on mitochondrial proteins and enzymes including arginase were observed. The 48 kDa polypeptide of mitochondrial fraction, that showed KCl-dependent alteration matched with Myb binding protein and 30 kDa bands resembles to arginase after MALDI MS/MS analysis. Results indicate KCl-dependent ultrastructural changes in mitochondria and release of mitochondrial arginase. The proposed membrane bound mitochondrial arginase could be mitochondrial arginase-II or altered form of cytosolic arginase-I contributing to KCl-induced arginase activity in H. fossilis.     

Purification and characterization of Helicobacter pylori arginase, RocF: unique features among the arginase superfamily.

The urea cycle enzyme arginase (EC 3.5.3.1) hydrolyzes l-arginine to l-ornithine and urea. Mammalian arginases require manganese, have a highly alkaline pH optimum and are resistant to reducing agents. The gastric human pathogen, Helicobacter pylori, also has a complete urea cycle and contains the rocF gene encoding arginase (RocF), which is involved in the pathogenesis of H. pylori infection. Its arginase is specifically involved in acid resistance and inhibits host nitric oxide production. The rocF gene was found to confer arginase activity to Escherichia coli; disruption of plasmid-borne rocF abolished arginase activity. A translationally fused His(6)-RocF was purified from E. coli under nondenaturing conditions and had catalytic activity. Remarkably, the purified enzyme had an acidic pH optimum of 6.1. Both purified arginase and arginase-containing H. pylori extracts exhibited optimal catalytic activity with cobalt as a metal cofactor; manganese and nickel were significantly less efficient in catalyzing the hydrolysis of arginine. Viable H. pylori or E. coli containing rocF had significantly more arginase activity when grown with cobalt in the culture medium than when grown with manganese or no divalent metal. His(6)-RocF arginase activity was inhibited by low concentrations of reducing agents. Antibodies raised to purified His(6)-RocF reacted with both H. pylori and E. coli extracts containing arginase, but not with extracts from rocF mutants of H. pylori or E. coli lacking the rocF gene. The results indicate that H. pylori RocF is necessary and sufficient for arginase activity and has unparalleled features among the arginase superfamily, which may reflect the unique gastric ecological niche of this organism.     

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 Mn²⁺ 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 MnCl₂. Enzyme activity in both fractions, in the absence of 15 mM MnCl₂, 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.     

Immunochemical characterization of human red cell acid phosphatase isozymes

An immunological study was performed on human red cell acid phosphatase (ACP1) isozymes encoded by different alleles, each of which is expressed as an electrophoretically fast (f) isozyme and a slow (s) isozyme. These isozymes reacted as two immunochemically different groups. Allele-specific reactions were not detected between either the f isozymes or the s isozymes. Quantitation of ACP1 isozymes in red cells by crossed immunoelectrophoresis revealed a phenotype-dependent variation in the concentration of isozyme protein. A simple gene dosage effect was indicated and the ordering of the ACP1 alleles (ACP1*A < ACP1*B < ACP1*C < ACP1*E) was identical to that found for enzyme activity levels. Also, an allele effect on the proportion between s and f isozymes (s/f) was observed; the ordering here was ACP1* B < ACP1*A < ACP1*, which is the same as that reported for the susceptibility to modulation with purines. These variations in isozyme protein levels appear to account for the phenotypic differences in the intensity of the isozyme bands, when activity-stained after electrophoresis, and in the red cell enzyme activity levels. Investigation of two carriers of a Null allele showed no evidence of an aberrant protein product, and half-normal concentrations of enzyme protein were observed in the red cells of these individuals.     

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.     

Genetic control of alkaline phosphatase isozymes in chicken duodenum

A genetic control of alkaline phosphatase (AP) in chicken duodenum was studied in a White Plymouth Rock strain. Unpurified chicken duodenum AP heated in an extraction procedure comprised either F or S band by isozyme types. On the other hand, chromatographically purified intestinal AP (NBCo, USA) had three bands, i.e., F', S' and B' bands. Characterization by urea, heat and neuraminidase treatments suggested that the genetic control of plasma AP isozymes may be applicable to the duodenum AP isozymes.     

Properties of arginase from liver of Macaca fascicularis: Comparison of normals with red blood cell arginase deficient monkeys

Deficiency of arginase (E.C. 3.5.3.1), the fifth enzyme of the urea cycle, was found in the red blood cells (RBCs) of Macaca fascicularis monkeys (<0.2 µmol arginine cleaved/g Hb/min; normal =49.2). Liver biopsies were obtained from two of these monkeys and from one monkey with normal levels of RBC arginase activity. Arginase from both groups of animals required Mn²⁺ for maximal enzyme activity and demonstrated a pH optimum of 10.2 in vitro. The activity of arginase in the livers of all three monkeys was 1.1 mmol arginine cleaved/g protein/min. The apparent K _m for arginine of arginase in the livers of the RBC-deficient monkeys was 7.4 and 5.9mm and in the normal monkey was 6.9mm. Similar patterns of heat denaturation were seen at 69 C without Mn²⁺ present and 79 C in the presence of 20mm Mn²⁺. No difference in mobility on either acidic or basic polyacrylamide gels for liver arginase from either RBC-deficient or normal monkeys was found. In addition, liver arginase from all three monkeys reacted similarly with anti-human liver arginase antibody. Liver arginases in RBC-deficient and normal monkeys were identical by ten criteria. These studies do not distinguish among several hypotheses for the genetic determination of arginase in different organs of this species and of man.     

Use of bead cellulose derivatives to isolation of bacterial alkaline proteinase by column liquid chromatography

Crude preparation of bacterial proteinase was purified by liquid chromatography. Combinations of individual ion-exchange chromatography methods and ion-exchange, hydrophobic and dye-ligand affinity chromatography, respectively, were used. The adsorbents were in all cases bead cellulose derivatives (Perloza), either commercially available (DEAE- and CM-Ostsorb) or prepared in the laboratory. Increase in column size resulted in a better separation efficiency of DEAE-Ostsorb IEC,i.e. step used in both separation protocols. The preparation of alkaline proteinase purified exclusively by this IEC method was highly active and comprised only trace amount of other proteins. This was proved by size-exclusion chromatography using the FPLC and HPLC mode. The relative molar mass of the enzyme (29.7 kDa) determined by SDS-polyacrylamide gel electrophoresis and its isoelectric point (pI 8.3) assayed by isoelectric focusing are at limit values typical for bacterial alkaline proteinases (30 kDa, pI about 9). The pH optimum of about 10.5 is typical for alkaline proteinase activity.     

Characterization of human carbonic anhydrase III from skeletal muscle

A third form of human carbonic anhydrase (CA III), found at high concentrations in skeletal muscle, has been purified and characterized. This isozyme shows relatively poor hydratase and esterase activities compared to the red cell isozymes, CA I and CA II, but is similar to these isozymes in subunit structure (monomer) and molecular size (28,000). CA III is liable to posttranslational modification by thiol group interaction. Monomeric secondary isozymes, sensitive to beta-mercaptoethanol, are found in both crude and purified material and can be generated in vitro by the addition of thiol reagents. Active dimeric isozymes, generated apparently by the formation of intermolecular disulfide bridges, also occur but account for only a small proportion of the total protein and appear only when the concentration of CA III is particularly high.     

Inhibitor profiles of alkaline phosphatases in bovine preattachment embryos and adult tissues.

The alkaline phosphatases are a small family of isozymes. Bovine preattachment embryos transcribe mRNA for two tissue-specific alkaline phosphatases (TSAP2 and TSAP3) beginning at the 4- and 8-cell stages. Whereas no mRNA has been detected in oocytes, there is maternally inherited alkaline phosphatase activity. It is not known which isozyme(s) is responsible for the maternal activity or when TSAP2 and TSAP3 form functional protein. No antibodies are available that recognize the relevant bovine alkaline phosphatases. Therefore, sensitivity to heat and chemical inhibition was used to separate the different isozymes. By screening tissues, it was determined that the bovine tissue-nonspecific alkaline phosphatase (TNAP) is inactivated by low temperatures (65C) and low concentrations of levamisole (<1 mM), whereas bovine tissue-specific isozymes require higher temperatures (90C) and levamisole concentrations (>5 mM). Inhibition by L-homoarginine and L-phenylalanine was less informative. Cumulus cells transcribe two isozymes and the pattern of inhibition suggested heterodimer formation. Inhibition of alkaline phosphatase in bovine embryos before the 8-cell stage indicated the presence of only TNAP. At the 16-cell stage the pattern was consistent with TNAP plus TSAP2 or -3 activity, and in morulae and blastocysts the pattern indicated that the maternal TNAP is fully supplanted by TSAP2 or TSAP3.     

Mercury Analysis of Acid- and Alkaline-Reduced Biological Samples: Identification of meta-Cinnabar as the Major Biotransformed Compound in Algae

The biotransformation of Hg^II in pH-controlled and aerated algal cultures was investigated. Previous researchers have observed losses in Hg detection in vitro with the addition of cysteine under acid reduction conditions in the presence of SnCl₂. They proposed that this was the effect of Hg-thiol complexing. The present study found that cysteine-Hg, protein and nonprotein thiol chelates, and nucleoside chelates of Hg were all fully detectable under acid reduction conditions without previous digestion. Furthermore, organic (R-Hg) mercury compounds could not be detected under either the acid or alkaline reduction conditions, and only β-HgS was detected under alkaline and not under acid SnCl₂ reduction conditions. The blue-green alga Limnothrix planctonica biotransformed the bulk of Hg^II applied as HgCl₂ into a form with the analytical properties of β-HgS. Similar results were obtained for the eukaryotic alga Selenastrum minutum. No evidence for the synthesis of organomercurials such as CH₃Hg⁺ was obtained from analysis of either airstream or biomass samples under the aerobic conditions of the study. An analytical procedure that involved both acid and alkaline reduction was developed. It provides the first selective method for the determination of β-HgS in biological samples. Under aerobic conditions, Hg^II is biotransformed mainly into β-HgS (meta-cinnabar), and this occurs in both prokaryotic and eukaryotic algae. This has important implications with respect to identification of mercury species and cycling in aquatic habitats.     

Characterization of normal, glutathione-deficient and arginase-deficient sheep erythrocytes by 1H-NMR spectroscopy

Normal sheep erythrocytes as well as glutathione- (GSH-) deficient and arginase-deficient sheep erythrocytes have been characterized by 1H nuclear magnetic resonance spectroscopy. The GSH deficiency is a result of defective amino acid transport (lesion 1), diminished gamma-glutamylcysteine synthetase activity (lesion 2), or both (lesions (1 + 2)). 1H-NMR spectra of normal sheep erythrocytes are similar to those for human erythrocytes, and consist of resonances from a number of small intracellular molecules, including GSH. In contrast, the resonances for GSH in the GSH-deficient erythrocytes are much weaker, and strong resonances are observed for lysine, threonine and ornithine or arginine, depending on the arginase activity, in erythrocytes with lesion 1 and lesions (1 + 2). A comparison of the intensity of GSH resonances in spectra for normal and GSH-deficient erythrocytes with GSH levels determined spectrophotometrically following reaction with the nonspecific thiol reagent 5,5'-dithiobis(2-nitrobenzoate) (DTNB) indicates that either not all of the GSH determined with Ellman's reagent is free and observable by 1H-NMR or that not all of the thiol determined by Ellman's reagent is GSH. If the latter is the case, the GSH levels determined with Ellman's reagent for erythrocytes with lesions (1 + 2) are most affected, which might account for their high susceptibility to oxidative stress.     

Amino acid metabolizing enzymes in rat submaxillary gland, normal or neoplastic, and in pancreas.

The activities of 12 enzymes, many related to ornithine metabolism, were measured in rat submaxillary gland, submaxillary gland tumors and pancreas. In submaxillary gland, the activities of arginase, ornithine aminotransferase, pyrroline-5-carboxylate reductase and glutamine synthetase were high, but no ornithine transcarbamylase or proline oxidase could be detected. In the fetal submaxillary gland, arginase was at almost adult levels while ornithine aminotransferase reached 50% of its adult value postnatally. Submaxillary tumors deviated from their cognate tissue by lower levels of amino acid metabolizing enzymes and by high concentrations of thymidine kinase. In pancreas, none of the pyrroline-5-carboxylate metabolizing enzymes were as high as in either liver or submaxillary gland. The outstanding activities were those of gamma-glutamyl transpeptidase and glutamate dehydrogenase. Although arginase activities in submaxillary gland and pancreas were quantitatively similar, they differed qualitatively: submaxillary gland contained the same variant as liver while the pancreatic isozymes resembled those of other nonhepatic tissues.