Ornithine Transcarbamylase Activity Research Articles

Isolation of an Aspergillus terreus mutant impaired in arginine biosynthesis and its complementation with the argB gene from Aspergillus nidulans.

Using filtration enrichment techniques, an Aspergillus terreus arginine auxotrophic strain which contains a mutation that abolishes ornithine transcarbamylase (OTCase) activity has been isolated. This mutant has been genetically transformed with the cloned Aspergillus nidulans OTCase gene. Prototrophic transformants arose at a frequency of about 50 transformants per microgram of plasmid DNA. Southern blot analysis of DNA from the transformants showed that the transforming DNA was ectopically integrated at different locations in the A. terreus genome, often in multiple tandem copies. The transformants were phenotypically stable for several mitotic divisions and retained their capacity to produce extracellular enzymes.

Zinc supplementation reduces blood ammonia and increases liver ornithine transcarbamylase activity in experimental cirrhosis.

Zinc deficiency is common in cirrhosis and may be involved in the alteration of ammonia metabolism. Rats with carbon tetrachloride-induced cirrhosis have high plasma ammonia and low serum and tissue zinc levels. We used this model to examine the effects of oral zinc supplementation on activities of plasma ammonia and liver ornithine transcarbamylase (a key enzyme in the urea cycle). These parameters were examined in two consecutive experiments. Each experiment included two groups of rats treated with carbon tetrachloride; one group received zinc in the drinking water during the induction of cirrhosis, and another served as a control group. Regardless of zinc supplementation, all carbon tetrachloride-treated rats exhibited similar micronodular cirrhosis, with similar histological appearance and liver function impairment. Cirrhotic rats without zinc supplementation showed high plasma ammonia and low serum and hepatic zinc levels and reduced liver ornithine transcarbamylase activity. Serum, hepatic zinc and liver ornithine transcarbamylase activity increased significantly in the zinc-supplemented group, and these rats' plasma ammonia levels became normal. Plasma ammonia level was significantly inversely correlated with liver ornithine transcarbamylase activity and positively correlated with serum and hepatic zinc content. Our results suggest that zinc deficiency may modify hepatic ornithine transcarbamylase activity and, therefore, ammonia disposal.

Hepatic glutamine metabolism in the septic rat.

1. The hepatic metabolism of glutamine, alanine, ammonia, urea, glutathione and glucose was studied in rats made septic by caecal ligation and puncture and was compared with that in rats that had undergone sham operation (laparotomy). 2. Sepsis resulted in increases in the plasma activities of gamma-glutamyltransferase (P less than 0.001), alanine aminotransferase (P less than 0.001) and aspartate aminotransferase (P less than 0.001), the serum total and direct bilirubin concentrations (P less than 0.001), and the blood lactate (P less than 0.01), glutamine (P less than 0.05), alanine (P less than 0.001) and urea (P less than 0.05) concentrations, but produced decreases in the blood ketone body (P less than 0.001) and glutathione (P less than 0.05) concentrations and in the plasma cholesterol concentration (P less than 0.05). These changes were associated with marked negative nitrogen balance in septic rats. 3. Sepsis increased total hepatic blood flow (by 22.7%) together with hepatic arterial flow (by 25.8%) and portal venous flow (by 18.7%). Sepsis resulted in marked increases in the net rates of hepatic extraction of glutamine (by 164%), alanine (by 138%) and ammonia (by 259%) with concomitant increases in the net rates of hepatic release of glutamate (by 105%), glutathione (by 87.5%), glucose (by 70.1%) and urea (by 100.4%). 4. Sepsis increased the activities of liver carbamoylphosphate synthase (by 16.4%), ornithine transcarbamylase (by 29.8%), argininosuccinate synthase (by 28.1%) and arginase (by 33.8%). 5. Septic rats exhibited marked increases in hepatic protein (by 46.0%), RNA (by 43.4%) and DNA (by 37.7%) contents. These changes were accompanied by marked increases in the activity of thymidine kinase (by 35.9%).(ABSTRACT TRUNCATED AT 250 WORDS)

X-chromosome inactivation in the liver of female heterozygous OTC-deficient Sparse-furash mice

X-chromosome inactivation patterns were investigated in livers of nine spfash female heterozygous ornithine transcarbamylase (OTC)-deficient mice. Quantitative morphometric analysis of cellular mosaicism was performed on sections of frozen liver reacted with purified anti-OTC antibody and prepared for immunofluorescent microscopy. Analysis of enzymatic OTC activity was performed on sections of these livers using a radiochromatographic technique. Several areas of cellular mosaicism were seen in each of the histological sections that were studied. The distribution of the volume fraction of the liver tissue cells having cells with normal OTC content among the nine mice ranged from 20 to 70% and it correlated (r = 0.8, P = 0.005) with the enzymatic activities of the respective livers. The extreme variegation of mosaic patches in the liver suggests the high probability that a single needle biopsy will be diagnostic in females heterozygous for an OTC mutation. This study also suggests that at the time of X inactivation, the number of primordial liver embryonic cells is small and the observed variegation of liver mosaicism probably results from complex migration patterns of liver cells during fetal development. This study shows that the spfash mouse is a suitable animal model for quantitative studies of X-chromosome inactivation in liver using immunohistochemical staining of OTC protein.

Correction of ornithine transcarbamylase (OTC) deficiency in spf-ash mice by introduction of rat OTC gene

We introduced rat ornithine transcarbumylase (OTC) gene into OTC-deficient spf-ash mice by mating spf-ash heterozygotes with transgenic mice which carried recombinant DNA composed of 1.3 kb of the 5′ flanking region of the gene fused onto rat OTC cDNA. The liver OTC activity of hemizygous spf-ash mice which carried the transgene was about twice that of nontransgenic spf-ash mice, and the small intestinal OTC activity was 6 times higher; the values being 12% and 27% of the control levels, respectively. The transgenic spf-ash mice showed normal hair growth without sparse, fur, nearly normalized urinary orotic acid excretion and normalized serum citrulline concentration.

Metabolic and secretory response of parotid cells to cationic amino acids. Uptake and catabolism of L-arginine and L-ornithine

L-Arginine and L-ornithine, which stimulate amylase release, are taken up by rat parotid cells. L-Arginine is converted, in an NADPH-dependent manner and to a limited extent to L-citrulline in parotid cell homogenates, despite the absence of ornithine transcarbamylase activity. L-Arginine is largely converted to urea and L-ornithine. The generation of putrescine and polyamines from L-ornithine occurs at a very low rate, relative to the cell content in preformed amines. The major fate of exogenous or arginine-derived ornithine consists in its conversion to L-glutamate, which is then further metabolized. These findings raise several hypotheses for the secretory response of the parotid cells to cationic amino acids, including their accumulation as positively charged molecules inside the cell and the generation of either NO, amines, substrates for a transglutaminase-catalyzed reaction, or ATP through oxidative catabolism. However, each of these hypotheses meets with objections, the modality for the stimulation of amylase release by cationic amino acids being eventually considered as an unsettled matter.

Late-onset ornithine transcarbamylase deficiency in male patients

We report on 21 male patients who presented after 28 days of age with ornithine transcarbamylase (OTC) deficiency, which we define as late-onset OTC deficiency. These patients appeared normal at birth, but irritability, vomiting, and lethargy, which were often episodic, later developed. The age at presentation ranged from 2 months to 44 years. Biochemical testing revealed hyperammonemia, hyperglutaminemia, hypocitrullinemia, increased urinary orotate excretion, and decreased liver OTC activity measured in vitro, which ranged from 0% to 15% of normal. Male patients who were older at presentation had a somewhat different pattern of presenting symptoms and were more likely to die. These data illustrate the phenotypic variability of OTC deficiency. Unexplained episodes of repetitive or protracted vomiting in association with progressive alterations in behavior or neurologic findings should suggest the diagnosis of a urea cycle defect (or another symptomatic inborn error of metabolism), regardless of the age or medical history of the patient.

Evaluation of the Transfer and Expression in Mice of an Enzyme-Encoding Gene Using a Human Adenovirus Vector

Mutant mice of the Spf-ash strain have an inherited defect in ornithine transcarbamylase (OTC) protein synthesis, and were used to ascertain the potential of recombinant adenoviruses for introducing and expressing the normal gene lacking in these mice. These OTC mutant mice are characterized by a reduction in the amount of OTC activity, resulting in hyperammonemia, pronounced orotic aciduria, growth retardation, and sparse fur until weaning. A recombinant adenovirus that harbors the rat OTC cDNA under the control of the viral major late promoter (MLP) was constructed and injected into such newborn mice. The effect of the virus was analyzed by monitoring the hepatic OTC enzyme during several months after the injection. An increase in OTC activity was detected and was accompanied by a diminution of orotic acid in the urine. The observation of MLP-OTC mRNA transcripts over 1 year following the injection attests to the relatively long-term presence of the transferred gene. In those mice showing the greatest OTC activity, a normalization of the fur was also observed. The experiments reported here document the feasibility of using adenovirus for the direct delivery in vivo of a gene to restore an impaired metabolism.

Complementation of the Aspergillus nidulans[formula omitted] mutation by ornithine transcarbamylase cDNA from rat liver

An Aspergillus nidulans strain which is deficient in ornithine transcarbamylase due to the arg B1 mutation was transformed with a plasmid containing the ornithine transcarbamylase cDNA from rat liver under the control of the amd S promoter. Stable transformants were obtained by selection on arginine free medium indicating complementation of the arg B mutation. Proof of expression of the rat enzyme in transformants was obtained by immunoprecipitation of all ornithine transcarbamylase activity from cell extracts with antibodies specific for the rat enzyme. The presence of catalytically active rat ornithine transcarbamylase in the transformants indicated that it is capable of being imported into mitochondria in A. nidulans, proteolytically processed and assembled into its homotrimeric form. In vitro uptake experiments using isolated A. nidulans mitochondria demonstrate that processing of the precursor of rat ornithine transcarbamylase occurs in two temporally separated steps as it does in rat liver mitochondria suggesting evolutionary conservation of the processing machinery. Up to 560 ng of active rat enzyme was produced per gm wet weight mycelia. Use of β-D-alanine, an inducer of amd S, as sole N-source resulted in increased levels of active rat ornithine transcarbamylase relative to uninduced cultures.

Urea cycle disorder in C3H-H-2° mice with juvenile steatosis of viscera

We determined the activities of urea cycle enzymes in the liver of C3H-H-2 o-jsv mice. The activities of all urea cycle enzymes decreased in the latter period of lactation. The activities of carbamylphosphate synthetase and ornithine transcarbamylase in some affected mice were undetectable. On the other hand, the activities of enzymes other than urea cycle enzymes were normal. We consider that the decrease in the urea cycle enzymes is caused by an abnormality in the mechanism of gene expression.

A case of ornithine transcarbamylase deficiency with acute and late onset simulating Reye's syndrome in an adult male.

A 15-year-old male was admitted to the hospital because of a disturbance of consciousness. He was in good mental and physical condition until the previous day, when he vomited and experienced a reduction of consciousness. He was brought to the hospital the day following the onset of symptoms. On admission the patient was stuporous. Neurological focal signs were not demonstrated. Computed tomographic (CT) scanning on admission showed no definite abnormality. Cerebrosprinal fluid had no pleocytosis. At this time, Reye's syndrome was suspected because of the acute change of consciousness and the presence of hyperammonemia. The disturbance of consciousness progressed and he became comatose on the day following admission. CT scanning showed that the suprasellar, ambient and quadrigeminal cisterns could almost not be detected and the bilateral ventricles were narrowed. The plasma aminogram demonstrated an elevation of alanine, lysine, glutamine, glutamic acid, aspartate and proline. Ketonuria was negative, but the urinary excretion of orotic acid was markedly increased. This data was indicative of the existence of an ornithine transcarbamylase (OTC) deficiency. The patient was died on the third day after hospitalization. The OTC activity in the liver was 10% of normal. Activities of other enzymes in the urea cycle were within normal limits. It is unusual that the symptoms of the enzyme deficiency occurred, both acutely and late at an adult age, as in this case. This case demonstrates that OTC deficiency should be considered in the differential diagnosis of hyperammonemia of adult onset.

An arginine to glutamine mutation in residue 109 of human ornithine transcarbamylase completely abolishes enzymatic activity in Cos1 cells.

Ornithine transcarbamylase (OTC) is an important enzyme in the detoxification of ammonia to urea, and its deficiency is the most common inborn error of ureagenesis in humans. Among 24 cases of OTC deficiency previously examined, three unrelated individuals all showed loss of a Taq I site in the OTC gene corresponding to codon 109, suggesting that this Taq I site may be prone to mutation. Two of these patients demonstrated the same C----T transition (in antisense strand) converting Arg109 to Gln. Although these studies implied a strong association between the missense mutation and OTC-deficient phenotype, a causal relationship could not be firmly established. We have investigated this relationship by reconstructing the mutation in vitro. A full-length human OTC cDNA was cloned into an SV40-based expression vector and has been reproducibly expressed at high levels in the cell line Cos1. By site-directed mutagenesis of this wild type sequence, we constructed a missense mutation which contains the C----T transition. Electroporation and transient assay in Cos1 indicated that the specific activity of mutant OTC was 100-fold lower than that of wild type. This result confirms that the Taq I alteration leading to the Gln missense is responsible for the OTC deficiency affecting the above patients.

Carbamoyl Phosphate Synthetase, Ornithine Transcarbamylase, and Aspartate Transcarbamylase Activities in the Pea Ovary

Carbamoyl phosphate synthetase (CPS), ornithine transcarbamylase (OTC), and aspartate transcarbamylase (ATC) were assayed in extracts from unpollinated ovaries of Pisum sativum L. CPS and OTC activities were, per milligram protein, the highest reported in a plant tissue, representing an estimated 0.1% of the protein in the ovary. The OTC/CPS and ATC/CPS ratios were about 100 and 0.5, respectively, indicating that most of the carbamoyl phosphate is used for arginine synthesis. The weight, protein content, and CPS, OTC, and ATC activities per ovary were determined during the senescence of the ovary and also during fruit set induced by treatment with gibberellic acid (GA(3)). In the nontreated ovary the weight and the protein first increased and then decreased dramatically, but the decrease in protein took place much earlier. In the GA(3)-treated ovaries the increase in weight was considerably greater than the increase in the protein. Whether or not the ovaries were treated with GA(3), CPS, OTC, and ATC activities closely followed the changes in protein, and thus their ratios and specific activities remained essentially constant. It appears that treatment with GA(3) increases the amount of protein and enzymic activities by preventing a large increase in the rate of protein degradation. In addition, the effects of acetylglutamate, ornithine, and UMP on CPS activity were studied. The pea enzyme exhibits regulatory properties intermediate between those of Escherichia coli and the ureotelic liver enzymes.

Carbamyl phosphate synthetase and ornithine transcarbamylase activities in enzyme-deficient human liver measured by radiochromatography and correlated with outcome.

Sensitive and specific radiochromatographic methods to measure enzymatic activities of carbamyl phosphate synthetase I (CPS I) and ornithine transcarbamylase (OTC) were developed. The activities of these enzymes were assayed in frozen liver tissue obtained from 23 individuals with hyperammonemia caused by CPS I (five patients) and OTC deficiency (18 patients). In addition, livers of one aborted fetus with OTC deficiency and four normal individuals were studied. The assays use radioactive ornithine as a substrate followed by separation of citrulline formed in the reactions by HPLC and quantitation of the radioactivity in both amino acids by a radioactivity flow monitor or by a scintillation counter. Both CPS I and OTC assays were linear with respect to length of incubation time and concentration of tissue homogenate. The sensitivity of the methods allowed measurements of CPS I and OTC activities as low as 0.1 mumol/g/min on 5 mg of liver tissue and the diagnosis of CPS I or OTC deficiency could be established on as low as 0.5 and 0.05 mg of tissue, respectively. CPS I activity in different sections of four normal livers was 3.01 +/- 0.16 mumol/g/min (mean +/- SEM, n = 19) and OTC activity was 93.4 +/- 6.3 (mean +/- SEM, n = 19). Residual enzymatic activity could be detected and measured in the liver tissues of one of the five subjects with CPS I deficiency and in 14 of 19 subjects with OTC deficiency. OTC/CPS I activity ratio in normal liver tissue was 31.2 +/- 1.3 (mean +/- SEM, n = 19), whereas this ratio ranged from 343 to greater than 5000 in CPS I deficient livers and from less than 0.02 to 1.55 in OTC deficient livers.(ABSTRACT TRUNCATED AT 250 WORDS)

The role of amino acids and their transport systems in the regulation of ureogenesis in the primary culture of adult rat hepatocytes.

The biosynthesis of urea, the metabolism of ammonia and the transport of amino acids were studied using the primary culture of hepatocytes from adult rats. The urea synthesis and ammonia detoxication were affected by the amino acids of urea cycle intermediates, such as ornithine, arginine and aspartate. When the hepatocytes were incubated in the medium containing 1 mM ammonium chloride, the transport activity of system-A, which was determined by the uptake of specific substrate methyl-2-amino isobutyric acid (MeAIB), did not change compared with the control level. However, the transport activity of ornithine was increased to a maximum after 4 hr of incubation with ammonia, and then decreased gradually to twice the control level. The activity of ornithine transcarbamylase (OTC) increased to twice the control. These results indicated that the amino acids of urea cycle intermediates, especially ornithine, can be the important regulators of ureogenesis.

Compared expression levels of ornithine transcarbamylase and carbamylphosphate synthetase in liver and small intestine of normal and mutant mice

Enzymatic assay, electrophoretic immunoblotting and RNA dot-blot techniques were employed to investigate the expression of the ornithine transcarbamylase (OTC) gene in liver and small intestine of Sparse fur mice with abnormal skin and hair (Spf-ash) and Sparse fur mice (Spf) which exhibit an X-linked OTC deficiency. We found a reduced OTC activity in these two tissues. We now show that this reduction is less pronounced in the intestine than in the liver of the Spf-ash strain. During the first 2 weeks of life, the deficiency appears to be less severe than in the adult mice. The enzymatic activity of carbamylphosphate synthetase I (CPS), another enzyme of the urea cycle, is significantly modified in the Spf mutant strain only.

Arginine dihydrolase pathway in Lactobacillus buchneri: a review

The arginine dihydrolase system was studied in homo- and hetero-fermentative lactic acid bacteria. This system is widely distributed in Betabacteria lactobacilli subgroup (group II in Bergey's Manual). It is generally absent in the Thermobacterium lactobacilli subgroup (group IA in Bergey's Manual) and also in the Streptobacterium subgroup (group IB in Bergey's Manual). It is present in some species of the genus Streptococcus (groups II, III and IV in Bergey's Manual). In Lactobacillus buchneri NCDO 110 the 3 enzymes of the arginine dihydrolase pathway, arginine deiminase, ornithine transcarbamylase and carbamate kinase, were purified and characterized. Arginine deiminase was partially purified (68-fold); ornithine transcarbamylase was also partially purified (14-fold), while carbamate kinase was purified to homogeneity. The apparent molecular weight of the enzymes was 199,000, 162,000 and 97,000 for arginine deiminase, ornithine transcarbamylase and carbamate kinase respectively. For arginine deiminase, maximum enzymatic activity was observed at 50°C and pH 6; for ornithine transcarbamylase it was observed at 35°C and pH 8.5, and for carbamate kinase at 30°C and pH 5.4. The activation energy of the reactions was determined. For arginine deiminase, △G ∗ values were: 8,700 cal mol −1 below 50°C and 380 cal mol −1 above 50°C; for ornithine transcarbamylase, the values were: 9,100 cal mol −1 below 35°C and 4,300 cal mol −1 above 35°C; for carbamate kinase, the activation energy was: 4,078 cal mol −1 for the reaction with Mn 2+ and 3,059 cal mol −1 for the reaction with Mg 2+. For characterization of the 3 enzymes, Km values for substrates and Ki values of inhibitors were determined. The results of kinetic analysis of ornithine transcarbamylase are consistent with a ping-pong mechanism. For carbamate kinase, the results of initial velocity studies have indicated that Mn 2+ ADP was as effective a substrate as Mg 2+ ADP; and product inhibition studies have shown that the enzyme has 2 distinct sites, one for nucleoside diphosphate and the other for carbamyl phosphate, and that its reaction with the substrates is of the random type. The formation of the arginine dihydrolase system in Lactobacillus buchneri NCDO 110 showed that the specific activities of arginine deiminase, ornithine transcarbamylase, and carbamate kinase were higher in galactose-grown cells than in glucose - or sucrose-grown cells. The 3 enzymes were induced by arginine, and this enzymatic system was not repressed by glucose.

Physiological analysis of bicozamycin high-producing Streptomyces griseoflavus used at industrial level.

Streptomyces griseoflavus, a bicozamycin-producing wild type strain and its high-producing one derived from it by multiple (greater than 15) mutagenic treatments, were analyzed physiologically and biochemically. The high-producing strain was characterized by: (1) An increased pool size of amino acids including leucine and isoleucine, precursors for bicozamycin synthesis, (2) an earlier and greater accumulation of intracellular ppGpp, (3) a more accentuated decrease in GTP pool size, (4) a higher specific activity of ornithine transcarbamylase which produces citrulline, (5) an increased ability to form aerial mycelium, and (6) an increased resistance to its own antibiotic. We propose that (1), (2) and (4) may be responsible for the high yields of bicozamycin and, possibly, of some other antibiotics produced by Streptomyces sp.

Induction of the five urea-cycle enzymes by glucagon in cultured foetal rat hepatocytes

Abstract Foetal hepatocytes obtained from rats at different stages were cultured in order to investigate the inducibility of the five urea-cycle enzymes by glucagon and dibutyryl cyclic AMP (Bt 2cAMP). When 18.5-day-old hepatocytes were cultured for 3 days with 10 –7 M glucagon, the activities of carbamoyl phosphate synthetase (CPS), argininosuccinase (ASL) and arginase were increased by 1.4-, 1.8- and 1.9-fold, respectively, as compared to controls. These effects were mimicked by 10 –4 M Bt 2cAMP, but the activities of ornithine transcarbamylase (OTC) and argininosuccinate synthetase (ASS) were never changed by the addition of these compounds. Hepatocytes cultured at earlier stages were not responsive to glucagon unless dexamethasone was added simultaneously, suggesting that this steroid might induce some steps necessary for glucagon action. Bt 2cAMP was effective as early as day 16.5 without requiring the presence of steroids. In addition, the effect of the cyclic nucleotide appeared additive or synergistic with that of dexamethasone. The simultaneous addition of actinomycin D did not affect the glucagon-induced increase in enzyme levels, thus suggesting a post-transcriptional effect of the hormone on the foetal enzyme activities. Insulin itself did not have any effect on the basal level of the enzyme activities and had only a moderate inhibitory effect on glucagon-induced ASL activity. This slight effect of insulin is in contrast with the marked inhibitory effect of dexamethasone on this enzyme activity that we described previously

Protection from N-nitrosodimethylamine-mediated liver damage by indole-3-carbinol

Indole-3-carbinol (I-3-C) was examined for its ability to protect mice against 24-hr N-nitrosodimethylamine (NDMA)-mediated hepatotoxicity. NDMA (20 mg/kg body weight) alone produced extensive hemorrhagic and centrolobular necrotic lesions, with a necrotic severity index of 3.0 ± 0.4 (scale of 0–5). Treatment with 50 mg/kg body weight of I-3-C by gavage, 1 hr prior to NDMA, substantially protected against hemorrhagic lesions. Furthermore, I-3-C lowered the NDMA-mediated tissue necrotic index to 1.5 ± 0.3, by reducing the extent of tissue necrosis rather than the severity in the necrotic region. Release of liver enzymes into the blood correlated with the histopathology; I-3-C reduced NDMA-mediated elevated activities of plasma alanine transaminase and ornithine transcarbamylase by 84 and 51.3%, respectively. Although no changes in nonprotein sulfhydryls were evident at 24-hr after NDMA, ascorbate levels were reduced to 40% of control values. However, treatment with I-3-C prior to NDMA prevented the decline in tissue ascorbate concentrations. In vitro, I-3-C was found to be a type II ligand for cytochrome P-450, with a Ks value of 237 μM. However, if such binding occurs in vivo, it does not protect against the approximately 60% decrease in hepatic cytochrome P-450 or the 80% decrease in NDMA demethylase I activity produced by NDMA. Since I-3-C slightly enhances cytochrome P-450 content and NDMA demethylase activity, the histopathologic protection by I-3-C must be due to factors other than inhibiting metabolic activation of NDMA.