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.     

Cell-free synthesis and transport of precursors of mutant ornithine carbamoyltransferases into mitochondria

Synthesis, mitochondrial transport and processing of ornithine carbamoyltrasferase (EC 2.1.3.3) were studied in mutant mice strains (sparse-fur, spf, and sparse-fur with abnormal skin and hair, spf-ash) which exhibit a deficiency in this enzyme. Spf mice have an increased amount (about 150% of control) of the enzyme with abnormal kinetic properties, whereas spf-ash mice have a decreased amount (about 10% of control) of the enzyme with apparently normal kinetic properties. Precursors of the mutant enzymes were synthesized in a reticulocyte lysate cell-free system. The hepatic level of translatable mRNA coding for the enzyme and the rate of the enzyme synthesis in liver slices of spf mice were 58 and 60% of the controls, respectively. In the case of spf-ash mice the activity of translatable mRNA for the enzyme was 10% of the controls. These results indicate that the decreased amount of ornithine carbamoyltransferase protein in spf-ash mice is due mainly to a decreased level of translatable mRNA for the enzyme, whereas the increase in the enzyme amount in spf mice is presumably the result of a decreased rate of enzyme degradation. The subunit molecular weight of the spf enzyme precursor was practically the same as that of the normal enzyme precursor (M_r 40 000). Both precursors synthetized in vitro could be taken up and processed similary to an apparently mature form (M_r 37 000). In the case of spf-ash enzyme, two discrete in vitro products were observed on sodium dodecyl sulfate polyacrylamide gel; one comigrated with the normal enzyme precursor and the other moved slightly slower. Both products appeared to be taken up and processed to the mature form of the enzyme.     

Immortalization of normal liver functions in cell culture: rat hepatocyte-hepatoma cell hybrids expressing ornithine carbamoyltransferase activity.

Normal rat hepatocytes have been fused with highly differentiated rat hepatoma cells. Some of the hybrids express a physiologically significant level of activity of the urea cycle enzyme ornithine carbamoyltransferase (OCT), a liver-specific function not found in the hepatoma cells. These hybrids have 10% of the adult rat liver OCT specific activity, incorporate 3H-ornithine into protein arginine, and can be selectively grown in arginine-free medium supplemented with ornithine. Somatic cell hybridization of normal differentiated cells with highly differentiated neoplastic cells of the same tissue type may be useful as a general method for obtaining permanent cell lines with new tissue-specific phenotypes.     

Altered enzyme activities and citrulline synthesis in liver mitochondria from ornithine carbamoyltransferase-deficient sparse-furash mice.

Male mice carrying the spfash mutation have 5-10% of the normal activity of ornithine carbamoyltransferase, yet are only slightly hyperammonaemic and develop quite well. A study of liver mitochondria from normal and spfash males showed that they differ in important ways. (1) The spfash liver contains about 33% more mitochondrial protein per g than does normal liver. (2) The specific activities of carbamoyl-phosphate synthetase (ammonia) and glutamate dehydrogenase are about 15% lower than normal in mitochondria from spfash mice, whereas those of beta-hydroxybutyrate dehydrogenase and cytochrome oxidase are 22% higher and 30% lower respectively. (3) In the presence of 10 mM-ornithine and the substrates for carbamoyl phosphate synthesis, coupled and uncoupled mitochondria from spfash mice synthesize citrulline at unexpectedly high rates, about 25 and 44 nmol/min per mg respectively. Though these are somewhat lower than the corresponding rates obtained with normal mitochondria, the difference does not arise from the deficiency in ornithine carbamoyltransferase, but from the lower carbamoyl-phosphate synthetase activity of the mutant mitochondria. (4) At lower external [ornithine] (less than 2 mM), a smaller fraction of the carbamoyl phosphate synthesized is converted into citrulline in spfash than in normal mitochondria. These studies show that what appears to be a single mutation brings about major adaptations in the mitochondrial component of liver. In addition, they clarify the role of ornithine transport and of protein-protein interactions in citrulline synthesis in normal mitochondria.     

A specific requirement for biotin in the synthesis of ornithine carbamoyltransferase by yeast

1. Growth of a biotin-requiring strain of Saccharomyces cerevisiae in a medium containing a suboptimum concentration of biotin for growth caused a decreased synthesis of ornithine carbamoyltransferase as compared with yeast grown in a medium containing an optimum concentration of biotin. Inclusion of the biotin homologues norbiotin or homobiotin, but not bishomobiotin, in the biotin-deficient medium caused an appreciable increase in ornithine carbamoyltransferase synthesis without affecting growth or synthesis of total RNA and protein. The addition of norbiotin to biotin-deficient medium had no effect on the respiratory activity of the yeast or on the synthesis of aspartate carbamoyltransferase, acid phosphatase, beta-fructofuranosidase or malate dehydrogenase. 2. Synthesis of acetylornithine deacetylase and acetylornithine acetyltransferase was slightly diminished by the imposition of biotin deficiency, but the effect was not as great as on ornithine carbamoyltransferase synthesis. Incorporation of norbiotin in the biotin-deficient medium had no marked effect on the synthesis of any other arginine-pathway enzyme except ornithine carbamoyltransferase. 3. l-Ornithine induced synthesis of ornithine carbamoyltransferase in yeast grown in biotin-deficient medium, but in yeast grown in this medium supplemented with norbiotin it repressed synthesis of the enzyme. l-Arginine had no detectable effect on ornithine carbamoyltransferase synthesis by the yeast grown in biotin-deficient medium with or without norbiotin. l-Aspartate repressed synthesis of ornithine carbamoyltransferase in biotin-deficient yeast and completely nullified the stimulatory effect of norbiotin on synthesis of the enzyme in this yeast. 4. There was no increase in ornithine carbamoyltransferase synthesis in biotin-deficient yeast incubated in phosphate buffer, pH4.5, containing glucose and biotin or norbiotin. In biotin-deficient yeast suspended in complete medium containing an optimum concentration of biotin, there was an increase in ornithine carbamoyltransferase synthesis only after the onset of growth.     

Phaseolotoxin-insensitive ornithine carbamoyltransferase of Pseudomonas syringae pv. phaseolicola: basis for immunity to phaseolotoxin.

Cell-free extracts from phaseolotoxin-producing strains of Pseudomonas syringae pv. phaseolicola grown at 18 degrees C, the optimum temperature for phaseolotoxin production, contain an ornithine carbamoyltransferase activity that is insensitive to phaseolotoxin. Extracts from the same strains grown at 30 degrees C, a temperature at which little or no detectable phaseolotoxin is produced, and from phaseolotoxin-nonproducing strains contain a phaseolotoxin-sensitive ornithine carbamoyltransferase activity. The phaseolotoxin-insensitive ornithine carbamoyltransferase activity is also less senstive to N delta-(phosphonacetyl)-L-ornithine than the phaseolotoxin-sensitive ornithine carbamoyltransferase activity of the corresponding strain.     

Citrulline synthesis in rat tissues and liver content of carbamoyl phosphate and ornithine

Rat liver ornithine carbamoyltransferase appears to be located exclusively in the mitochondria; the activity that is found in the soluble fraction is indistinguishable from mitochondrial ornithine carbamoyltransferase by simple kinetic criteria, and seems to result from breakage of mitochondria during homogenization. Of several rat tissues studied, only the liver and the mucosa of small intestine contain significant amounts of ornithine carbamoyltransferase; the activity in intestinal mucosa is less than one thousandth of that in liver. Qualitatively, this distribution coincides with that of carbamoyl phosphate synthetase I and its cofactor, acetylglutamate. The rat liver contents of carbamoyl phosphate and ornithine were 0.1 and 0.15mumol/g wet wt. of tissue respectively. On the basis of these values, it is proposed that in vivo the ornithine carbamoyltransferase activity of liver may be much lower than its maximal activity in vitro might suggest.     

Efficient mitochondrial import of newly synthesized ornithine transcarbamylase (OTC) and correction of secondary metabolic alterations in spf(ash) mice following gene therapy of OTC deficiency.

BACKGROUND: The mouse strain sparse fur with abnormal skin and hair (spf(ash)) is a model for the human ornithine transcarbamylase (OTC) deficiency, an X-linked inherited urea cycle disorder. The spf(ash) mouse carries a single base-pair mutation in the OTC gene that leads to the production of OTC enzyme at 10% of the normal level. MATERIALS AND METHODS: Recombinant adenoviruses carrying either mouse (Ad.mOTC) or human (Ad.hOTC) OTC cDNA were injected intravenously into the spf(ash) mice. Expression of OTC enzyme precursor and its translocation to mitochondria in the vector-transduced hepatocytes were analyzed on an ultrastructural level. Liver OTC activity and mitochondrial OTC concentration were significantly increased (300% of normal) in mice treated with Ad.mOTC and were moderately increased in mice receiving Ad.hOTC (34% of normal). The concentration and subcellular location of OTC and associated enzymes were studied by electron microscope immunolocalization and quantitative morphometry. RESULTS: Cytosolic OTC concentration remained unchanged in Ad.mOTC-injected mice but was significantly increased in mice receiving Ad.hOTC, suggesting a block of mitochondria translocation for the human OTC precursor. Mitochondrial ATPase subunit c [ATPase(c)] was significantly reduced and mitochondrial carbamy delta phosphate synthetase I (CPSI) was significantly elevated in spf(ash) mice relative to C3H. In Ad.mOTC-treated mice, the hepatic mitochondrial concentration of ATPase(c) was completely normalized and the CPSI concentration was partially corrected. CONCLUSIONS: Taken together, we conclude that newly synthesized mouse OTC enzyme was efficiently imported into mitochondria following vector-mediated gene delivery in spf(ash) mice, correcting secondary metabolic alterations.     

Complementary expression of glutamine synthetase and carbamoylphosphate synthetase I in ornithine carbamoyltransferase-deficient mouse liver (spf–ash mouse)

 Glutamine synthetase and carbamoylphosphate synthetase I expression was examined immunohistochemically in livers of spf–ash homozygous and hemizygous mice, in which one of the urea cycle enzymes (ornithine carbamoyltransferase) is deficient and hyperammonemic disorders are obvious. In the mutant adult mouse liver, only hepatocytes lining central veins expressed glutamine synthetase. In contrast, other hepatocytes expressed carbamoylphosphate synthetase I but not glutamine synthetase. This complementary expression pattern is similar to that seen in wild-type mouse liver. In the liver of mutant young mice, which showed severe retarded growth and abnormal hair and skin development, the developmental expression pattern of both enzymes was also similar to that of the corresponding wild-type liver. However, suppression of carbamoylphosphate synthetase I expression in the pericentral hepatocytes occurred later in the mutant than in wild-type liver. These results show that high plasma concentrations of ammonium ions, which are one of the substrates for both the enzymes, do not change their complementary expression. Instead they support the idea that factor(s) associated with central veins rather than humoral factors direct pericentral hepatocytes to express glutamine synthetase and to suppress carbamoylphosphate synthetase I expression. Accepted: 24 April 1997     

Genetic and physiological characterization of Pseudomonas aeruginosa mutants affected in the catabolic ornithine carbamoyltransferase.

In Pseudomonas aeruginosa arginine can be degraded by the arginine "dihydrolase" system, consisting of arginine deiminase, catabolic ornithine carbamoyltransferase, and carbamate kinase. Mutants of P. aeruginosa strain PAO affected in the structural gene (arcB) of the catabolic ornithine carbamoyltransferase were isolated. Firt, and argF mutation (i.e., a block in the anabolic ornithine carbamoyltransferase) was suppressed specifically by a mutationally altered catabolic ornithine carbamoyltransferase capable of functioning in the anabolic direction. The suppressor locus arcB (Su) was mapped by transduction between hisII and argA. Second, mutants having lost suppressor activity were obtained. The Su- mutations were very closely linked to arcB (Su) and caused strongly reduced ornithine carbamoyltransferase activities in vitro. Under aerobic conditions, a mutant (PA0630) which had less than 1% of the wild-type catabolic ornithine carbamoyltransferase activity grew on arginine as the only carbon and nitrogen source, at the wild-type growth rate. When oxygen was limiting, strain PA0630 grown on arginine excreted citrulline in the stationary growth phase. These observations suggest that during aerobic growth arginine is not degraded exclusively via the dihydrolase pathway.     

Anabolic ornithine carbamoyltransferase of Escherichia coli and catabolic ornithine carbamoyltransferase of Pseudomonas putida. Steady-state kinetic analysis.

The anabolic and catabolic ornithine carbamoyltransferases of Pseudomonas putida display an undirectional catalytic specialization: in citrulline synthesis for the anabolic enzyme, in citrulline phosphorolysis for the catabolic one. The irreversibility of the anabolic enzyme in vitro has been previously explained by its kinetic properties, whereas the irreversibility of the catabolic transferase in vivo was shown to be due to its allosteric behaviour. In this work a steady-state kinetic analysis has been carried out on the catabolic ornithine carbamoyltransferase at pH 6.8 in the presence of the allosteric activator, phosphate. The kinetic mechanism of Escherichia coli ornithine carbamoyltransferase serving as a reference was also determined. For the E. coli enzyme in the reverse direction, the initial velocity patterns converging on the abscissa were obtained with either citrulline or arsenate as variable substrate. The inhibition by the product ornithine was linear competitive with respect to citrulline and linear non-competitive with respect to arsenate. In the forward direction phosphate and its analogs induce an inhibition by ornithine which is partial and competitive with respect to carbamoylphosphate. Together with the results of thermo-inactivation studies in the presence of each reactant, this observation suggests a random kinetic mechanism, but with most of the reaction flux following the path where carbamoylphosphate adds before ornithine, when substrates are present at Km levels. The allosteric catabolic ornithine carbamoyltransferase of Pseudomonas displays qualitatively the same pattern as the E. coli enzyme.     

Correction of mouse ornithine transcarbamylase deficiency by gene transfer into the germ line

The sparse fur with abnormal skin and hair (Spf-ash) mouse is a model for the human X-linked hereditary disorder, ornithine transcarbamylase (OTC) deficiency. In Spf-ash mice, both OTC mRNA and enzyme activity are 5% of control values resulting in hyperammonemia, pronounced orotic aciduria and an abnormal phenotype characterized by growth retardation and sparse fur. Using microinjection, we introduced a construction containing rat OTC cDNA linked to the SV40 early promoter into fertilized eggs of Spf-ash mice. The expression of the transgene resulted in the development of a transgenic mouse whose phenotype and orotic acid excretion are fully normalized. Thus, the possibility of correcting hereditary enzymatic defect by gene transfer of heterologous cDNA coding for the normal enzyme has been demonstrated.     

Regulation of arginine biosynthesis in Saccharomyces cerevisiae: isolation of a cis-dominant, constitutive mutant for ornithine carbamoyltransferase synthesis.

A cis-dominant mutation linked to argF, the structural gene specifying ornithine carbamoyltransferase, and affecting the control of the synthesis of this enzyme has been obtained. The level of ornithine carbamoyltransferase in this mutation is depressed and less repressible by addition of L-arginine than it is in the wild-type strain. Of 38 tetrads analyzed, resulting from a cross of a strain harboring this mutation with a strain carrying an argF- mutation, none was a tetratype or a nonparental ditype. This operator mutation helps to define a negative mode of control of the synthesis of the arginine biosynthetic enzymes, as had been suggested earlier upon the isolation of argRI- (arg80), argRII- (arg81), and argRIII- (arg82) specific regulatory mutations.     

Measurement of the number of ornithine decarboxylase molecules in rat and mouse tissues under various physiological conditions by binding of radiolabelled α-difluoromethylornithine

The binding of alpha-difluoromethylornithine, an irreversible inhibitor, to ornithine decarboxylase was used to investigate the amount of enzyme present in rat liver under various conditions and in mouse kidney after treatment with androgens. Maximal binding of the drug occurred on incubation of the tissue extract for 60min with 3mum-difluoromethyl[5-(14)C]ornithine in the presence of pyridoxal phosphate. Under these conditions, only one protein became labelled, and this corresponded to ornithine decarboxylase, having M(r) about 100000 and subunit M(r) about 55000. Treatment of rats with thioacetamide or carbon tetrachloride or by partial hepatectomy produced substantial increases in ornithine decarboxylase activity and parallel increases in the amount of enzyme protein as determined by the extent of binding of difluoromethyl[5-(14)C]ornithine. Similarly, treatment with cycloheximide or 1,3-diaminopropane greatly decreased both the enzyme activity and the amount of difluoromethyl-[5-(14)C]ornithine bound to protein. In all cases, the ratio of drug bound to activity was 26fmol/unit, where 1 unit corresponds to 1nmol of substrate decarboxylated in 30min. These results indicate that even after maximal induction of the enzyme in rat liver there is only about 1ng of enzyme present per mg of protein. When mice were treated with androgens there was a substantial increase in renal ornithine decarboxylase activity, the magnitude of which depended on the strain. There was an excellent correspondence between the amount of activity present and the capacity to bind labelled alpha-difluoromethylornithine in the mouse kidney extracts, but in this case the ratio of drug bound to activity was 14fmol/unit, suggesting that the mouse enzyme has a higher catalytic-centre activity. After androgen induction, the mouse kidney extracts contain about 170ng of enzyme/mg of protein. These results indicate that titration with alpha-difluoromethylornithine provides a valuable method by which to quantify the amount of active ornithine decarboxylase present in mammalian tissues, and that the androgen-treated mouse kidney is a much better source for purification of the enzyme than is rat liver.     

Urea cycle enzymes in human liver: ontogenesis and interaction with the synthesis of pyrimidines and polyamines

Summary All the five enzymes of urea synthesis and the formation of urea in vitro can already be demonstrated in human liver as early as the 9th week of fetal development. At this stage the activity of carbamoyl phosphate synthetase is the highest, whereas that of ornithine carbamoyltransferase is the lowest as compared to those in the adult. The kinetic parameters of the urea cycle enzymes are the same in fetal liver as in adult liver, except that the Km values of ornithine carbamoyltransferase for L-ornithine are 3.5 mM and 0.42 mM in the fetus and in adult liver, respectively.Urea formation in vivo seems to begin in the second half of fetal life, and a gradual increase can be detected in the activity of the enzymes of urea synthesis. The activity of ortnithine decarboxylase, the glutamine-dependent carbamoyl phosphate synthetase and aspartate carbamoyltransferase, however, changes in the opposite direction.The concentration of carbamoyl phosphate and aspartate remains constant, but that of ornithine gradually decreases during ontogenesis. The ornithine, carbamoyl phosphate and aspartate pools are probably utilized in the polyamine, pyrimidine and urea syntheses at varying rates.     

X-chromosome inactivation in human liver: confirmation of X-linkage of ornithine transcarbamylase.

Histochemical assay for ornithine transcarbamylase (OTC) activity in fixed frozen hepatic sections from a woman heterozygous for OTC deficiency revealed two populations of hepatocytes: those with normal activity and those with no activity. This observation, in conjunction with data from previous family studies, confirms the hypothesis that the gene for OTC is X-linked. It also provides the first cytologic demonstration of cellular mosaicism for a liver-specific cell product.     

Function of arginase in lactating mammary gland

The potential for a considerable formation of ornithine exists in lactating mammary gland because of its arginase content. Late in lactation arginase reaches an activity in the gland higher than that present in any rat tissue except liver. Occurrence of the urea cycle can be excluded since two enzymes for the further reaction of ornithine in the cycle, carbamoyl phosphate synthetase I and ornithine carbamoyltransferase, are both absent from this tissue. Instead, carbamoyl phosphate synthetase II appears early in lactation, associated with accumulation of aspartate carbamoyltransferase and DNA, consistent with the proposed role of these enzymes in pyrimidine synthesis. The facts require another physiological role for arginase apart from its known function in the urea cycle. Significant activity of ornithine aminotransferase develops in mammary gland in close parallel with the arginase. By this reaction, ornithine can be converted into glutamic semialdehyde and subsequently into proline. The enzymic composition of the lactating mammary gland is therefore appropriate for the major conversion of arginine into proline that is known to occur in the intact gland.     

Evidence of carbamoylphosphate induced conformational changes upon binding to human ornithine carbamoyltransferase.

Human liver ornithine carbamoyltransferase undergoes absorbance changes in the UV region upon formation of the carbamoylphosphate-norvaline-enzyme ternary complex. The UV changes are similar in the presence of carbamoylphosphate alone, whilst they are lower in the presence of ornithine or norvaline alone. The extent of the UV changes correlates with the enzyme susceptibility to proteolytic degradation. The free native enzyme is completely and rapidly hydrolyzed by trypsin, whilst it is partially protected upon carbamoylphosphate binding. The extent of protection increases for the carbamoylphosphate-norvaline-enzyme ternary complex. These results strongly suggest that the binding of the first substrate, i.e. carbamoylphosphate, to human ornithine carbamoyltransferase induces a large protein isomerization, which regards the polar domain plus a part of equatorial domain of each subunit.     

Strategies to Rescue the Consequences of Inducible Arginase-1 Deficiency in Mice

Arginase-1 catalyzes the conversion of arginine to ornithine and urea, which is the final step of the urea cycle used to remove excess ammonia from the body. Arginase-1 deficiency leads to hyperargininemia in mice and man with severe lethal consequences in the former and progressive neurological impairment to varying degrees in the latter. In a tamoxifen-induced arginase-1 deficient mouse model, mice succumb to the enzyme deficiency within 2 weeks after inducing the knockout and retain <2 % enzyme in the liver. Standard clinical care regimens for arginase-1 deficiency (low-protein diet, the nitrogen-scavenging drug sodium phenylbutyrate, ornithine supplementation) either failed to extend lifespan (ornithine) or only minimally prolonged lifespan (maximum 8 days with low-protein diet and drug). A conditional, tamoxifen-inducible arginase-1 transgenic mouse strain expressing the enzyme from the Rosa26 locus modestly extended lifespan of neonatal mice, but not that of 4-week old mice, when crossed to the inducible arginase-1 knockout mouse strain. Delivery of an arginase-1/enhanced green fluorescent fusion construct by adeno-associated viral delivery (rh10 serotype with a strong cytomegalovirus-chicken β-actin hybrid promoter) rescued about 30% of male mice with lifespan prolongation to at least 6 months, extensive hepatic expression and restoration of significant enzyme activity in liver. In contrast, a vector of the AAV8 serotype driven by the thyroxine-binding globulin promoter led to weaker liver expression and did not rescue arginase-1 deficient mice to any great extent. Since the induced arginase-1 deficient mouse model displays a much more severe phenotype when compared to human arginase-1 deficiency, these studies reveal that it may be feasible with gene therapy strategies to correct the various manifestations of the disorder and they provide optimism for future clinical studies.     

A fatal variant of human ornithine carbamoyltransferase is stimulated by Mg2+

Biochemical studies of a female who died at 2 years of age from a possible genetic variant of ornithine carbamoyltransferase (OCTase) deficiency are reported. The patient had severe psychomotor retardation with plasma ammonia levels throughout life reaching as high as 500 mumole/liter. The average OCTase level in the patient's liver was 2% of that in normal livers. Preincubation with 0.05 M MgCl2 resulted in a 570% increase in OCTase activity (13% of control). Citrate synthase and carbamoyl-phosphate synthase I were present at essentially normal levels. Unusual Mg2+ requirements have not been recognized in previous reports of OCTase deficiency, suggesting a genetic variant in this patient.