Carbamyl Phosphate synthetase-I Research Articles

Protein Restriction Specifically Decreases the Abundance of Serum Albumin and Transthyretin Nuclear Transcripts in Rat Liver

The expression of the genes for serum albumin and several other plasma proteins is decreased in animals consuming inadequate amounts of dietary protein. To define the specificity of this phenomenon, we examined the effect of dietary protein restriction on the abundance of the mRNA for nine genes in rat liver. The results of this and previous studies indicate that genes in liver can be divided into two classes based on their response to protein restriction. Group I genes (albumin, transthyretin, carbamyl phosphate synthetase-I, class I alcohol dehydrogenase, insulin-like growth factor-I) exhibit decreased expression in response to protein restriction. In contrast, the expression of group II genes (hypoxanthine-guanine phosphoribosyl transferase, ubiquitin, H-ferritin, insulin-like growth factor binding proteins-1, -2 and -4) is either unchanged or increased in response to protein restriction. To investigate the molecular mechanism(s) leading to the decreased level of albumin and transthyretin mRNA in protein-restricted animals, the effect of protein restriction on the abundance of albumin and transthyretin nuclear transcripts was examined. The results demonstrated that protein restriction specifically decreased the abundance of albumin and transthyretin nuclear transcripts, indicating that the reduction in mRNA levels is caused at least partly by a decrease in gene transcription.

Effect of amino acid limitation on the expression of 19 genes in rat hepatoma cells

We showed previously that the abundance of serum albumin mRNA is decreased in H4-II-E rat hepatoma cells limited for a single essential amino acid (phenylalanine, methionine, leucine, or tryptophan). To define the specificity of this phenomenon, we examined the effect of amino acid limitation on the abundance of mRNAs for 19 genes in the H4-II-E cells. These genes included six genes whose expression is either completely liver-specific or highly enriched in the liver compared with other tissues [albumin, transthyretin (TTR), transferrin, carbamyl phosphate synthetase-I, urate oxidase, class I alcohol dehydrogenase], as well as a number of ubiquitously expressed "housekeeping" genes. The results indicated that the 19 genes could be divided into three classes based on their response to amino acid limitation. Class I genes (the six liver-specific genes and alpha-tubulin) exhibit decreased expression in response to amino acid limitation. The expression of class II genes [beta 2-microglobulin, hypoxanthine-guanine phosphoribosyl transferase (HPRT), H-ferritin, ubiquitin (UbB), insulin-like growth factor binding protein-4, HNF-1 alpha] is not significantly affected by amino acid limitation. Class III genes [gadd153, beta-actin, ubiquitin (UbC), phosphoglycerate kinase-1, C/EBP alpha, C/EBP beta] exhibit increased expression in response to amino acid limitation. Thus, specific inductive as well as repressive effects on gene expression are quite common in amino acid-limited cells. The observation that all six genes whose expression is liver-specific exhibited decreased expression in amino acid-limited cells suggests a common mode of regulation of these genes by amino acid availability. The strong induction by amino acid limitation of the C/EBP inhibitor gadd153 is of interest in this regard, as increased levels of gadd153 could interfere with C/EBP, which is required for high expression of most liver-specific genes. To investigate further the molecular mechanism for the decrease in albumin mRNA abundance, albumin nuclear transcript levels were quantified in control and tryptophan-limited cells. Tryptophan limitation caused a decrease in albumin nuclear transcript abundance, and this decrease preceded the decrease in albumin mRNA, suggesting that the decrease in albumin mRNA was caused at least partly by a decrease in albumin gene transcription. Additional experiments with actinomycin D indicated that albumin mRNA was also destabilized in the tryptophan-limited cells. Thus, the overall results indicate that the decrease in albumin mRNA in the tryptophan-limited cells is caused by a specific decrease in albumin nuclear transcript abundance and destabilization of albumin mRNA.

Cloning and sequence of a cDNA encoding human carbamyl phosphate synthetase I: molecular analysis of hyperammonemia

Carbamyl phosphate synthetase I (CPSI) is the first enzyme involved in urea synthesis. CPSI deficiency is an autosomal recessive disorder characterized by hyperammonemic coma in the neonatal period. To analyze the enzyme and gene structures, and to elucidate the nature of mutations in CPSI deficiency, we isolated cDNA clones encoding human liver CPSI. Oligo(dT)-primed and random primer human liver cDNA libraries in λgt11 were screened using 5', middle, and 3' fragments of the rat CPSI cDNA as probes. Seven positive clones covered the full-length cDNA sequence with an open reading frame encoding a precursor polypeptide of 1500 amino acids (aa) (deduced M r, 164828) with a putative N-terminal presequence of 38 or 39 aa, a 5'-untranslated sequence of 118 bp and a 3'-untranslated sequence of 597 bp. Comparison with the rat CPSI cDNA showed that the deduced aa sequence of the human liver CPSI precursor is 94.4% identical to the rat enzyme precursor. A molecular analysis was made of the genomic DNA from three patients with CPSI deficiency. Heterogeneity of hybridized fragments that may or may not be the cause of the deficiency was apparent on the DNA blots from tissues from one patient.

BWTG3 hepatoma cells can acquire phenylalanine hydroxylase, cystathionine synthase and CPS-I without genetic manipulation, but activation of the silent OTC gene requires cell fusion with hepatocytes

The mouse hepatoma BWTG3 has been tested for its ability to grow in three different media that select for traits normally expressed in adult liver: homocysteine medium to select for cystathionine synthase (CS), tyrosine-free medium for phenylalanine hydroxylase (PH), and ornithine medium for carbamylphosphate synthetase-I (CPS-I) and ornithine transcarbamylase (OTC). In no case were the cells immediately capable of bulk growth, showing that all these traits were in some degree deficient. However, the cultures in homocysteine medium and in tyrosine-free medium both gave rise, spontaneously, to growing clones with frequencies of approximately 10(-3) and 10(-5), respectively. The deficiencies of CS and PH were accordingly excluded from further study, in view of their inherent instability. In contrast, no colonies ever formed in ornithine medium. Though neither CPS-I nor OTC were detectable in stock BWTG3 cells, it was found that CPS-I was readily inducible by hormones. The deficiency of OTC, however, appeared to be totally stable showing no reversion in response either to hormones or to azacytidine treatment. This deficiency was investigated by fusing the hepatoma to OTC+ liver cells prepared from normal or sparse-fur (spf) mice. Sparse-fur mice were used because their OTC is mutant and has a distinctive pH-dependence. OTC+ hybrids were readily produced, without the need for any specific selection for OTC, and, in one case at least, with only minimal chromosome segregation. In all the OTC+ hybrids made with spf cells, there was clear reactivation of the wild-type, hepatoma-derived OTC gene.(ABSTRACT TRUNCATED AT 250 WORDS)

A case of carbamylphosphate synthetase-I deficiency associated with secondary carnitine deficiency —l-carnitine treatment of CPS-I deficiency

We describe a male infant with congenital hyperammonaemia due to partial carbamylphosphate synthetase-I (CPS-I) deficiency. At 21 days of age, he had convulsions and at 53 days of age hyperammonaemic coma. Therapy with sodium benzoate, L-arginine, essential amino acids, L-carnitine and peritoneal dialysis lowered the blood ammonia levels, and his clinical manifestations improved. The CPS-I activity in liver tissue obtained by open biopsy was about 25.6% of normal values. The serum and urine free carnitine levels in the patient decreased during the hyperammonaemic crisis and were low at 7 months of age. After oral administration of L-carnitine (10 mg/kg per day) at 7 months of age, the mean blood ammonia levels decreased significantly, accompanied by an increase in serum and urine free carnitine levels. We propose the use of L-carnitine therapy to prevent secondary carnitine deficiency in patients with CPS-I deficiency as well as ornithine transcarbamylase (OTC) deficiency.

Activity of the carbamyl phosphate synthetase I promoter in liver nuclear extracts is dependent on a cis-acting C/EBP recognition element.

We have identified an essential cis element in the proximal promoter region of the rat carbamyl phosphate synthetase I (CPSI) gene that is requisite for promoter activity in liver nuclear extracts. Excess synthetic oligonucleotides specifying this region abolished promoter-dependent in vitro transcription. We show that C/EBP, a nuclear factor enriched in liver but found as well in other tissues, such as gut, fat, and lung, interacts with an inverted repeat, GTTGCAAC, at the core of the essential cis element. In brain, a tissue that did not express CPSI or contain significant levels of C/EBP, a different factor was capable of binding at or near the C/EBP recognition element. Activity of the CPSI promoter in liver nuclear extracts was also dependent on sequences 5' to the C/EBP motif; presumably, factors binding to elements within this upstream region are instrumental in restricting CPSI gene expression to liver and intestinal mucosa.

Activity of the Carbamyl Phosphate Synthetase I Promoter in Liver Nuclear Extracts Is Dependent on a cis-Acting C/EBP Recognition Element

We have identified an essential cis element in the proximal promoter region of the rat carbamyl phosphate synthetase I (CPSI) gene that is requisite for promoter activity in liver nuclear extracts. Excess synthetic oligonucleotides specifying this region abolished promoter-dependent in vitro transcription. We show that C/EBP, a nuclear factor enriched in liver but found as well in other tissues, such as gut, fat, and lung, interacts with an inverted repeat, GTTGCAAC, at the core of the essential cis element. In brain, a tissue that did not express CPSI or contain significant levels of C/EBP, a different factor was capable of binding at or near the C/EBP recognition element. Activity of the CPSI promoter in liver nuclear extracts was also dependent on sequences 5' to the C/EBP motif; presumably, factors binding to elements within this upstream region are instrumental in restricting CPSI gene expression to liver and intestinal mucosa.

The role of hepatic ornithine transcarbamylase deficiency in the orotic aciduria of pregnant mice

Groups of normal and heterozygote sparse-fur (spf) mutant mice were studied at various stages of gestation, to assess the effects of normal pregnancy on orotate excretion, hepatic mitochondrial urea cycle enzymes and any predisposition to the development of fatty liver. Results show a higher total daily excretion of urinary orotate by normal pregnant mice on the 8th and 15th days of gestation, which came to within the usual basal range of excretion of non-pregnant mutant heterozygotes with hereditary ornithine transcarbamylase deficiency. Liver ornithine transcarbamylase and carbamyl phosphate synthetase-I activities were reduced in pregnant mice on the 16th day of gestation ( P < 0.05). No fatty change, bile stasis or glycogen depletion was discernible on optical microscopy in normal or mutant mice. Nonspecific changes were seen on ultrastructural examination. Orotic aciduria seen in pregnant mice may be directly related to a physiological deficiency of liver ornithine transcarbamylase. However, the depletion of both the mitochondrial urea cycle enzymes, seen on the 16th day of pregnancy, may be indicative of a metabolic stress at the mitochondrial level.

Hepatotoxicity of sodium valproate in ornithine trasncarbamylase-deficient mice

Susceptibility to sodium valproate (SV) hepatotoxicity was investigated in male sparse-fur mutant (spf/Y) mice with X-linked ornithine transcarbamylase (OTC) deficiency, as compared to normals (+/Y). SV was given in drinking water, in increasing concentrations of 0, 0.05, 0.15 and 0.25%. Actual SV intake was similar in both groups. There were no significant changes in orotate excretion, but α-amino nitrogen increased progressively with SV intake in both groups. Valproate-treated animals also had a significant increase in hepatic carbamyl phosphate synthetase- I (CPS-I) activity. OTC-deficient spf/Y mice showed 33% mortality and morbidity at 0.05–0.15% valproate, while normal mice remained non-symptomatic. spf/Y Mice also showed a higher incidence of hepatocellular necrosis, microvesicular steatosis and polymorphic infiltration. Centrilobular necrosis was seen only in symptomatic OTC-deficient mice, indicating an idiosyncratic hepatotoxic response which may be different from the dose-related effects seen in all SV-treated mice. Electron microscopy of liver sections from severely affected spf/Y mice showed marked abnormalities of mitochondria, which appeared swollen or rounded. The rough endoplasmic reticulum was dilated and filled with a flocculent material. It is postulated that the idiosyncratic response in OTC-deficient mice may be caused by an interaction between a metabolic aberration of mitochondria and toxic metabolites of valproate.

Avian and Mammalian Mitochondrial Ammonia-Detoxifying Systems in Tortoise Liver

Liver mitochondria from the desert tortoise Gopherus agassizii and the Texas tortoise G. berlandieri contain glutamine synthetase, the enzyme used by birds and higher reptiles to detoxify ammonia generated intramitochondrially during amino acid catabolism. Tortoise mitochondria also contain carbamyl phosphate synthetase-I and ornithine transcarbamylase, the enzymes used by mammals for this purpose. Since chelonid reptiles have changed little since their divergence from the stem reptiles, this finding suggests that both systems were present in the latter before the emergence of the ruling reptile, avian, and mammalian lines of descent.

The transport and processing of carbamyl phosphate synthetase-I in mouse hepatic mitochondria

Carbamyl phosphate synthetase-I (CPS-I) 2 2 Abbreviations used: CPS-I, carbamyl phosphate synthetase-I; pCPS-I, precursor of carbamyl phosphate synthetase-I; EDTA, Disodium ethylene diamine tetraacetate; CCCP, carbamyl cyanide m-chlorophenyl hydrazone; SDS, sodium dodecyl sulfate; PMSF, phenylmethyl sulfonyl fluoride; TLCK, N α-p-tosyl-L-lysine chloromethyl ketone; TPCK, L-1-p-Tosylamino-2-phenylethylchloromethyl ketone , purified from mouse hepatic mitochondria consists of electrophoretically homogeneous polypeptide species of 160 kilodaltons molecular weight (Kd). Monospecific antibody to CPS-I immunoprecipitated a putative precursor of 165Kd protein from in vitro translation products programmed with mouse liver free polysomes or poly(A) RNA. Isolated mitochondrial particles can take up and process pCPS-I into mature CPS-I of 160Kd in an in vitro transport system. The in vitro transport of CPS-I is energy dependent and requires intact mitochondria. The processing of pCPS-I appears to involve a single endoproteolytic cleavage.

Cell-free translation and thyroxine induction of carbamyl phosphate synthetase I messenger RNA in tadpole liver.

Total RNA of tadpole and frog (Rana catesbeiana) liver was isolated by either 7 or 8 M guanidine . HCl extraction and translated in a cell-free protein-synthesizing system derived from rabbit reticulocytes. The identity of carbamyl phosphate synthetase I[carbamoyl-phosphate synthase (ammonia); ATP:carbamate phosphotransferase (dephosphorylating), EC 2.7.2.5] synthesized in vitro with the purified enzyme was established as follows: (i) immunoprecipitation by a specific antibody; (i) comigration with purified carrier enzyme on sodium dodecyl sulfate/polyacrylamide gel electrophoresis; (iii) copurification with carrier enzyme by affinity chromatography on Cibacron Blue F3GA-coupled agarose; and (iv) formation of identical proteolytic cleavage products. Inclusion of protease inhibitors in the system resulted in no apparent change in the polypeptide molecular weight. These results indicate that carbamyl phosphate synthetase I is synthesized as a polypeptide that is indistinguishable from the mature enzyme by the analytical methods used and that it is not grossly modified during its transport into mitochondria. The level of translatable mRNA for carbamyl phosphate synthetase-I in tadpole liver was increased about 2-fold 1 day after thyroxine treatment and did not change significantly through 4 subsequent days of treatment. Thus the thyroxine-induced synthesis of carbamyl phosphate synthetase I in tadpole liver is at least partly due to an increase of translatable mRNA for this enzyme.

Postnatal Changes in Carbamylphosphate Synthetase-I and Ornithine Transcarbamylase Activities after Normal Birth, Premature Delivery and Prolonged Gestation in Rat Liver: Effects of Actinomycin D and Glucose

The development of two urea cycle enzymes, carbamylphosphate synthetase-I and ornithine transcarbamylase was examined in neonatal rat liver. Normal birth on day 21.5 caused a marked increase of both activities as early as 4 h of extrauterine life. That increase occurred later in newborns delivered by cesarian section on day 21.5, but they exhibited a greater hepatic urea level. Premature delivery on day 20.5 caused a marked increase of both activities and hepatic urea level while prolonged gestation abolished these increases. The postnatal increase in both activities and in hepatic urea level was therefore dependent on a factor associated with birth. The in vivo administration of actinomycin D (2 microgram) at birth did not abolish the postnatal increase of both activities. Moreover, the administration of glucose (25 mg every 2 h) to newborns delivered by cesarian section on day 21.5 abolished the postnatal increase of ornithine transcarbamylase activity and hepatic urea level 23 h later. It seems that the transient hypoglycemia and appearance of gluconeogenesis at birth were the physiological mechanisms involved in the postnatal induction of ornithine transcarbamylase activity.

Preparation of crystalline carbamyl phosphate synthetase-I from frog liver.

Ammonia- and N-acetylglutamate-dependent carbamyl phosphate synthetase-I (EC 2.7.2.5), the mitchondrial enzyme involved in the initial step of urea biosynthesis, was purified to homogeneity from frog liver and crystallized. The purification involved extraction of a particulate fraction with cetyltrimethylammonium bromide in the presence of the protease inhibitors antipain, leupeptin, chymostatin, and pepstatin; acetone precipitation; and affinity chromatography with Cibacron blue F3GA-coupled agarose. The enzyme was adsorbed to the gel at pH 8.3 in the presence of 5 mM MgCl2 and eluted with magnesoum-free buffer. The enzyme crystallized as either elongated, thin, rectangular plates or as clusters of small crystals from 37 to 40% saturated ammonium sulfate. The enzyme moved as a single polypeptide band on sodium dodecyl sulfate/polyacrylamide gel electrophoresis with a molecular weight of 160,000. In the absence of protease inhibitors, proteolysis of the enzyme occurred with the formation of an enzymatically active fragment with a subunit molecular weight of 139,000.

Carbamyl phosphate synthesis in invertebrates

1.1. Carbamyl phosphate synthesis in mitochondria from land snail (Otala lactea and Helix aspersa) hepatopancreas, earthworm (Lumbricus terrestris) gut tissue and land planarian (Bipalium kewense) whole body is mediated, in part, by an enzyme that utilizes l-glutamine as a substrate and requires N-acetyl-l-glutamate as a cofactor. This glutamine enzyme was first described in hepato-pancreas of the snail Strophocheilus oblongus and now appears to be widely distributed among invertebrates.2.2. In addition to the glutamine enzyme, mitochondria from the earthworm and land planarian and possibly the snail Helix also possess an enzyme that utilizes ammonia in the presence of N-acetyl-l-glutamate and is therefore similar to carbamyl phosphate synthetase-I, the enzyme for arginine and urea biosynthesis in vertebrates.3.3. In all four species, the major site of carbamyl phosphate synthesis was in the mitochondrion and the major enzyme detected was the glutamine enzyme. In the earthworm, the amount of carbamyl phosphate synthetase-I was only slightly less than that of the glutamine enzyme and both enzymes were found in the soluble cellular fraction as well as in the mitochondrial fraction during localization studies.