Ornithine Carbamyl Transferase Research Articles

Regulation of Arginine Metabolism in Germinating Peanut ( Arachis hypogea L.) Seeds

Summary Regulation of arginase (EC 3.5.3.1), ornithine ketoacid aminotransferase (EC 2.6.1.13), and ornithine carbamyltransferase (EC 2.1.3.3) was investigated by measuring the specific activities of these enzymes in the cotyledons and the embryo axis of germinating peanut ( Arachis hypogea L.) seeds grown under various conditions. It was observed that the activities of arginase and ornithine ketoacid aminotransferase of peanut cotyledons and embryo axis were reduced by auxins, ethrel, and ethylene chlorohydrin at early stages of germination. In the cotyledons, the activities of these enzymes returned to the control level as seedling growth progressed. However, in case of the embryo axis, the activities of these enzymes increased to more than the control levels. Exogenous polyamines increased the activities of these enzymes in the embryo axis and this encrease in the activities was prevented by cycloheximide. The activity of ornithine carbamyltransferase of the cotyledons and the embyo axis was not affected by any of these compounds. That the regulatory control on the embryo axis arginase and ornithine ketoacid aminotransferase was not influenced by the accompanying cotyledons, was demonstrated by cultivating the embryo axis in a synthetic medium.

Normal serum activities of liver enzymes in Swedish paint industry workers with heavy exposure to organic solvents.

The serum activities of the liver enzymes alanine aminotransferase, aspartate aminotransferase, ornithine carbamyl transferase, and gamma-glutamyl transferase were examined in 47 paint industry workers and unexposed age matched referents. The workers were exposed to a mixture of industrial solvents, of which xylene was the main component in most cases. The median total exposure was about 50% of Swedish 1981 threshold limit values according to measurements of individual solvent exposure performed at the same time. No differences in enzyme activities were shown either when the whole exposed and referent groups were compared or when the five workers with outstanding solvent exposures of five times the TLV or more were compared with their referents. It is concluded that in most workers the liver seems to remain largely undamaged from inhalation exposure to a commonly used mixture of non-chlorinated solvents. In many workers this seems to hold true even for high exposures for limited periods.

The precursor to ornithine carbamyl transferase is transported to mitochondria as a 5S complex containing an import factor

The precursor to ornithine carbamyl transferase (Mr = 40,000) was synthesized in a rabbit reticulocyte lysate system and purified by immunoaffinity chromatography. Import of purified precursor by isolated mitochondria depended upon the presence of import factor(s) in fresh reticulocyte lysate. Velocity sedimentation analyses indicated that import factor binds to precursor to form a 5S complex (∼ 90 kDa); in this form, precursor was efficiently imported by isolated mitochondria. The ability of the 5S complex to deliver precursor into mitochondria was not affected by pretreatment with high concentrations of RNase. Import factor did not bind to mitochondria in the absence of precursor; upon binding of precursor to mitochondria in the presence of import factor, subsequent transmembrane uptake of precursor did not require the continued presence of additional lysate components.

Membrane effects of various drugs on isolated rat hepatocytes and erythrocytes

The relationship between hepatotoxicity and membrane effects of clinically used drugs on erythrocytes was investigated. The cytotoxicity of various drugs to isolated rat hepatocytes was determined by the leakage of glutamic oxalacetic transaminase (GOT) and ornithine carbamyl transferase (OCT) into the surrounding medium. Inhibition of hypotonic hemolysis of rat erythrocytes was measured as the hemoglobin concentration in the supernatant solution. Drugs such as tricyclic antidepressants, local anesthetics (dibucaine), and bile acids (chenodeoxycholic acid), which have both a membrane stabilizing effect on erythrocytes at low concentrations and a hemolytic effect at higher concentrations, induced enzyme leakage from hepatocytes. The concentration at which hemolysis occurred corresponded to the concentration which caused a marked enzyme leakage from hepatocytes. These phenomena were observed for alkyltrimethylammonium salts (C 10 to C 16), the order of cytotoxicity to hepatocytes and erythrocytes was C 16 > C 14 > C 12 > C 10. Marked enzyme leakage was observed for chenodeoxycholic acid at 1 × 10 −3 m but not for ursodeoxycholic acid. The order of membrane stabilizing and lytic potency of tricyclic antidepressants was chlorimipramine > amitriptyline > desipramine > imipramine. These results suggest that these membrane effects of various drugs on erythrocytes may be useful for screening for hepatocytotoxicity in vitro.

Biochemical markers of chronic alcoholism

Usefulness of several biochemical markers for the monitoring of chronic alcoholism were studied. Among generally used markers, only γ-GTP showed a significant difference between alcoholic and non-alcoholic liver diseases. Serum glutamate dehydrogenase (GDH) activity was significantly high in alcoholic liver disease. When the ratios of GDH to ornithine carbamyl transferase (OCT) were calculated, differences between alcoholic and non-alcoholic liver diseases became clearer without overlapping of any value. Serum desialo-transferrin was found in about 60% of the alcoholics, and disappeared by abstinence. Microheterogeneity of serum protein was also found in other glycoproteins. Serum prealbumin level was significantly high in alcoholics without severe liver disease. Acetaldehyde dehydrogenase (ALDH) activity of erythrocytes was significantly low in alcoholics, and gradually increased after abstinence. These results indicate that microheterogeneity of glycoproteins, serum prealbumin level and erythrocyte ALDH activity are good markers of alcohol abuse, and serum GDH/OCT ratio is the most sensitive marker of alcoholic liver injury. Serum γ-GTP activity is a good marker of both conditions.

Differential import and processing of the precursors to F 1-ATPase β-subunit and ornithine carbamyltransferase by liver, spleen, heart and kidney mitochondria

The cytoplasmically made subunits 2 (β) and 3 (γ) of the H +-ATPase from mammalian mitochondria are synthesized in vitro as larger polypeptides. In contrast, pre-cytochrome c could not, on the basis of its molecular weight, be distinguished from the mature polypeptide. This was shown by programming a reticulocyte lysate with rat heart RNA and immunoprecipitating the labeled translation products with polypeptide-specific antibodies. When a translated lysate containing the precursor to the β-subunit was incubated with isolated rat spleen mitochondria, it was converted to the mature subunit and was no longer susceptible to externally added trypsin. The conversion to the mature form occurred in the absence of protein synthesis. This post-translational maturation process of the β-subunit was more efficient when carried out with spleen or liver mitochondria than with heart or kidney mitochondria. The converse relative efficiency was observed when the processing of the precursor to ornithine carbamyltransferase by these mitochondria was examined. These results indicate that mitochondria do not discriminate against tissue-specific mitochondrial proteins. In addition, the observed varying degrees of efficiency of mitochondria from different tissues in importing and processing these two precursors suggest that the activity of precursor(s)-specific translocation-maturation systems varies between different types of mitochondria.

Serum glutamate dehydrogenase and ornithine carbamyl transferase in Reye's syndrome.

Parallel increases in the ornithine carbamyl transferase and glutamate dehydrogenase activities were observed in the serum of 22 Reye's syndrome patients. The increase in the activity of glutamate dehydrogenase was masked by a dialyzable inhibitor. It is proposed that the measurement of serum ornithine carbamyl transferase and glutamate dehydrogenase activities may be useful as an indicator of liver mitochondrial damage in Reye's syndrome.

Neuropathology of ornithine carbamyl transferase deficiency.

Neuropathologic findings in two cases of ornithine carbamyl transferase (OCT) deficiency are presented. In one, a boy 3 days old, the only abnormality related to the enzyme defect was metabolic gliosis which was unusual in distribution being present mainly in the brain stem. In the other case, a girl who died at the age of 2 3/12 years, the brain showed metabolic gliosis in typical location, widespread ulegyria, and moderate atrophy of the internal granular layer in the cerebellum accompanied by development of expansions of Purkinje cell dendrites. Case 2 demonstrates how rapidly changes previously demonstrated in the cerebral cortex after a few months of illness can proceed to massive hemispheric destruction. By now ulegyria has been observed in several disorders of the urea cycle.

Ornithine carbamyl transferase in Reye's syndrome

Serum levels of ornithine carbamyl transferase activities were determined in the acutely ill and convalescent Reye's syndrome patients and in their parents. Acutely ill Reye's syndrome patients had elevated levels of serum ornithine carbamyl transferase activities as compared to those in controls. The convalescent Reye's syndrome patients and their parents had normal levels of serum ornithine carbamyl transferase activities. These results suggest that an inborn metabolic defect was not responsible for the increase in serum ornithine carbamyl transferase activities in Reye's syndrome.

Impact of larval trematode infection on the nitrogen excretion and ornithine-urea cycle enzymes of the pulmonate snail host, Lymnaea luteola

The freshwater gastropod Lymnaea luteola infected with xiphidiocercariae of Prosthogonimus sp. showed striking changes in nitrogen excretion. Infected snails excreted significantly less total Kjeldahl nitrogen into the amibient medium. A significant drop in urea nitrogen alone accounted for this drop in total nitrogen excreted, as there was no change in ammonia excretion. While no significant change was seen in the activity of ornithine carbamyltransferase and arginosuccinate lyase in infected snails, arginase activity invariably dropped. The present study thus has revealed that it is not the urea production that occurs in the digestive gland that is affected upon infection, but it is arginolysis that occurs in other tissues, like foot and mantle, that is curtailed.

Plasma ornithine carbamyl transferase level as an indicator of ischaemic injury of rat liver.

The activity of ornithine carbamyl transferase (OCT) and glutamate pyruvate transaminase (GPT) in serum has been correlated with the extent of necrosis 24 h after different periods of ischaemia in rat liver. The extent of necrosis has been quantified as the volume density of necrosis in the total ischaemic liver lobes using tetranitro BT. The GPT-activity in serum is maximal between 1 and 5 h after different periods of ischaemia, whereas OCT reaches its maximum between 5 and 12 h after ischaemia. The total amount of leaked enzyme-activity as well as the peak value give a linear correlation with the extent of necrosis for OCT and GPT. There is a difference between the character of these two enzymes in that a small leakage of GPT does not indicate liver cell necrosis later on. However, the appearance of OCT in the blood, an enzyme localized in the mitochondrial matrix, has a predictive value for the extent of necrosis, likely to occur later on. GPT, an enzyme from the cytoplasm, can also occur in the blood during the reversible stage of liver cell damage.

Fatal intoxication by 1,2-dichloroethane-a case report

This is a case report of acute fatal intoxication after accidental exposure to 1,2-dichloroethane in a 51-year-old man. Clinical manifestations, blood chemistry, and autopsy findings are described. High levels of lactate and ammonia in blood had been observed before the elevation of glutamic transaminases, lactic dehydrogenase, and creatine phosphokinase. Ornithine carbamyl transferase and glutamic oxaloacetic transaminase of mitochondrial origin (m-GOT) were remarkably high.

POSTNATAL ACCUMULATION OF ADENINE NUCLEOTIDES IN RABBIT LIVER MITOCHONDRIA AND THE DEVELOPMENT OF CITRULLINE SYNTHESIS

Liver mitochondria (mito) accumulate adenine nucleotides right after birth thereby greatly increasing the matrix ATP+ADP+AMP content (BBA: 681:300, 1982). Bioenergetic function is also enhanced. Overall, there is a large increase in matrix ATP that might be expected to stimulate ATP-dependent pathways within the mito. Carbamyl phosphate synthetase and ornithine carbamyltransferase are enzymes of the urea cycle that are localized in the matrix; they catalyze citrulline (CIT) synthesis from ornithine, NH3, and CO2 in an ATP-dependent reaction sequence. We investigated whether the postnatal increase in the matrix ATP pool size affects CIT synthesis. Mito were isolated from rabbit pups at birth and at 2 hrs of age. ATP, ADP and AMP were determined enzymatically in PCA extracts. CIT synthesis (assayed by 14CO2 fixation) increased between birth and 2 hrs, from 1.38±.35 to 2.40± .26 umol/hr/mg mito protein. The matrix ATP+ADP+AMP pool size (nmol/mg mito protein) increased from 5.7±.8 at birth to 15.1±1.6 at 2 hrs and the matrix ATP/ADP ratio increased from 1.47 to 3.52. The larger adenine nucleotide pool size together with the higher ATP/ADP ratio resulted in a 3.4-fold increase in matrix ATP concentration. Two moles ATP are required per mole CIT, so the increase in ATP appears to account for the 1.8-fold increase in CIT synthesis. Conclusion: The accumulation of adenine nucleotides and the maturation of energetic functions within the first few hours after birth allow a rapid postnatal increase in the capacity for CIT synthesis. (NIH HD 16936)

SALICYLATE INHIBITS CITRULLINE SYNTHESIS IN MITOCHONDRIA: IMPLICATIONS FOR REY'S SYNDROME

In Reye's Syndrome (RS) there is a general decrease in the specific activities of mitochondrial enzymes, including two of the urea cycle, carbamyl phosphate synthetass and ornithine carbamyl-transferase. These two enzymes catalyze the synthesis of citrul-line (CIT) from ornithine, NH3, and CO2; the functional impairment of this reaction sequence probably contributes to hyperammonemia in RS. Salicylate (SA) has been implicated as a synergistic factor in the pathogenesis of RS. In this study we investigated whether SA inhibits CIT synthesis in isolated rat liver mitochondria. CIT synthesis was assayed at 37°C by measuring 14CO2 fixation in the presence of ornithine and NH3 (ABB 178:19,1977). SA inhibited CIT formation (μmol/hr/mg mito protein) as follows:Gentisic acid, salicyluric acid and acetaminophen had no effect at 5 mM. CIT synthesis is dependent on matrix ATP: a mechanism of inhibition was suggested by further experiments showing that SA uncouples oxidative phosphorylation and decreases the rate of ATP synthesis. Matrix ATP/ADP ratios were decreased by SA as follows: 1.15±.04 (O SA); 0.90±.21 (0.25 mM SA); 0.63±.09 (0.5 mM SA); 0.11±.04 (1 mM SA). Conclusion: SA is a potent inhibitor of CIT synthesis at physiological concentrations, probably as a result of its uncoupling action which lowers matrix ATP. The implication for RS is that SA might inhibit urea cycle function beyond the already compromised level. (NIH 14936)

Experience with a Simple High-Performance Liquid Chromatography Method for the Analysis of Purine and Pyrimidine Nucleosides and Bases in Biological Fluids

Purine and pyrimidine nucleosides and bases were analysed using an isocratic reverse-phase HPLC technique and utilising simple sample preparation. The method has been successfully used to separate a large number of biologically occurring nucleic acid products within a 30-minute period and to identify them by their chromatographic and optical properties. A number of metabolic disorders have been identified, including hypoxanthine-guanine phosphoribosyl transferase and ornithine carbamyl transferase deficiencies. The method has been in routine use for 2 years performing both quantitative and qualitative analysis and has proved to be robust and reliable.

Assessment of functional, morphological, and enzymatic tests for acute nephrotoxicity induced by mercuric chloride.

The relative merits of a comprehensive series of contemporary methods for detection of acute nephrotoxicity were evaluated. Male Sprague-Dawley rats were given 0, 0.25, 0.5, 1.0, or 3.0 mg mercuric chloride (HgCl2)/kg body weight by ip injection. Indices of nephrotoxicity were examined 8, 24, 48, 72, and 96 h later. Alterations in urine osmolality, volume, and protein levels were seen within 24 h in response to 1 mg/kg or more of HgCl2. Administration of 0.5-3.0 mg/kg produced dose-dependent increases in urinary excretion of maltase activity and glucose by 24 h, the period of peak effect. There was no increase in maltase or alkaline phosphatase (AP) activity in the serum of these animals. Enzymuria was not apparent in rats that had marked elevations in serum AP, argininosuccinate lyase, and ornithine carbamyl transferase activities as a result of physical (i.e., dichlorodifluoromethane-frozen) or chemical (carbon tetrachloride-induced) damage of the liver. Morphological alterations, in the proximal tubular epithelium of perfusion-fixed kidneys from HgCl2-dosed rats, paralleled the changes in enzyme excretion with respect to time of onset and dose-effect. There was a dose-dependent inhibition of tetraethylammonium (TEA) and p-aminohippurate (PAH) uptake by renal cortical slices at 24 h. Interestingly, increases in uptake of TEA and PAH were seen 8 h after a 1-mg/kg dose. Clearance of inulin and PAH in vivo were altered at 8 h by 0.5 and 1 mg/kg. Marked depression of these functional indices was seen at 24 h, by which time blood urea nitrogen (BUN) levels were increased. The 0.5- and 1.0-mg/kg doses also produced time- and dose-dependent increases in intracellular Na+ content which were maximal at 24 h. These results illustrate the importance of using a combination of biochemical and functional tests to elucidate the sequence of events in the kidney following toxic insult. Nevertheless, some of the simpler, traditional techniques (e.g., histopathology, urinalyses, BUN) were sensitive and organ-specific, and should continue to be very useful in nephrotoxicity testing/screening.

MASKING BY ENZYME INHIBITOR OF RAISED SERUM GLUTAMATE DEHYDROGENASE ACTIVITY IN REYE'S SYNDROME

Serum glutamate dehydrogenase (GDH) activity was greatly raised (up to 830 times the upper limit of normal) in 16 patients with Reye's syndrome. The serum activity was masked by an inhibitor, and the rises were observed only after dialysis or sample dilution. Serum GDH values from 38 paediatric patients, including 10 with hyperammonaemia due to other causes, showed no such rise after dialysis. Only 1 of 13 adult patients with liver disease had high GDH activity, but this level was not increased after dialysis. Serum ornithine carbamyl transferase activity was also raised in patients with Reye's syndrome, but levels were not increased after dialysis. The ratio of dialysed/undialysed GDH activity clearly distinguished all Reye's patients from controls. The inhibition of a mitochondrial enzyme which regulates ammonia metabolism may contribute to the hyperammonaemia of Reye's syndrome. Serum GDH levels before and after dialysis would seem to be a useful diagnostic aid in Reye's disease.

The pathway of arginine catabolism in the parasitic flagellate Trichomonas vaginalis

Arginine is rapidly depleted from the medium during the cultivation of T. vaginalis in a defined or semi-defined medium. It is broken down to ornithine, ammonia and carbon dioxide by the three enzymes of the dihydrolase pathway: arginine deiminase, catabolic ornithine carbamyltransferase (OCTase) and carbamate kinase. Arginase and urease as well as citrulline hydrolase appear to be absent. Ornithine, a product of the pathway was further converted to putrescine by an active ornithine decarboxylase. Apparent substrate K m values determined were arginine deiminase, 103 μM; catabolic OCTase, 71 μM; ornithine decarboxylase 134 μM. A substrate level phosphorylation is associated with the pathway; the significance of this to the overall energy economy of the cell is unclear.

Membrane and cytosolic components affecting transport of the precursor for ornithine carbamyltransferase into mitochondria.

A higher molecular weight precursor (Mr = 39,000) to the liver mitochondrial matrix enzyme, ornithine carbamyltransferase (Mr = 36,000), is imported and processed by heart mitochondria in vitro in a manner similar to liver mitochondria. In both systems, however, an additional 37-kDa ornithine carbamyltransferase polypeptide appears, but this arises from nonspecific events and, therefore, does not represent a bona fide intermediate in the overall processing sequence. Our experiments demonstrate that the outer mitochondrial membrane of mitochondria contains a protease-sensitive (5 micrograms of trypsin or chymotrypsin/ml, 15 min at 2 degrees C), salt-resistant (1.0 M KCl) protein which is required to maintain import functions. In addition, functional post-translational import requires a component of the reticulocyte lysate (i.e. cytosol) that is used for initially synthesizing precursor enzyme. The component is retained by Sephadex G-25. Import of Sephadex G-25-excluded precursor is restored by fresh reticulocyte lysate but not by a combination of other additives, including Mg2+, K+, ATP, ADP, Pi, succinate, and total translation mixture (minus lysate).

Toxicity of venalot (a mixture of coumarin and troxerutin) in the baboon

Venalot, a mixture of coumarin and troxerutin, in the proportion 1 to 6 respectively, was given orally to baboons at dosages of 0, 100, 300 and 1000 mg kg day for 26 weeks. Vomiting, usually within 3 h of administration and considered to be of central origin, in addition to vomiting immediately after dosing, was noted in animals receiving 1000 mg kg day . At this level, collapse on several occasions in two animals, one of which died, was also observed. Another animal receiving 1000 mg kg day was killed for humane reasons following a period of weight loss, reduced appetite and deterioration in body condition. However, no adverse effect on body weight gain, food or water consumption, ophthalmoscopic or electrocardiographic examinations were noted in any other animals during this study. Increased levels of liver function (serum leucine amino-peptidase (LAP), and serum ornithine carbamyl transferase (OCT)) were noted during the dosing period, together with slightly increased liver weights terminally for animals receiving 1000 mg kg day ; however, as no morphological or ultrastructural changes were noted, these findings were considered to be attributable to hypertrophy.