Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:6.3.5.5 (CPS)
1,262 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

1. When NH3 was added to isolated rat-liver mitochondria incubated with succinate and bicarbonate, oxidation of succinate was stimulated to a greater extent than could be accounted for by the net formation of carbamoyl phosphate. 2. Measurement of the rate of incorporation of [14C]bicarbonate into carbamoyl phosphate, after the mitochondria had been preincubated with NH3 and unlabelled bicarbonate, revealed that flux through carbamoyl-phosphate synthetase (ammonia) was much greater than the net formation of carbamoyl phosphate indicated. 3. It is concluded that part of the carbamoyl phosphate produced in the absence of ornithine is degraded. About 20% of the degradation can be accounted for by non-enzymatic reactions of carbamoyl phosphate outside the mitochondria. It is proposed that the remainder of the degradation of carbamoyl phosphate occurs by partial reversal of the reaction of carbamoyl-phosphate synthetase.
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PMID:Activity of carbamoyl-phosphate synthetase (ammonia) in isolated rat-liver mitochondria: cycling of carbamoyl phosphate in the absence of ornithine. 708 32

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 ornithine 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, carbamoylphosphate and aspartate pools are probably utilized in the polyamine, pyrimidine and urea syntheses at varying rates.
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PMID:Urea cycle enzymes in human liver: ontogenesis and interaction with the synthesis of pyrimidines and polyamines. 708 58

Addition of ammonium ions to isolated rat hepatocytes stimulated the rate of synthesis of pyrimidines. Isolation and quantification of pyrimidine nucleotides orotic acid and the acid-hydrolyzed product of carbamoyl-aspartic acid by ion-exchange chromatography and high-pressure liquid chromatography show a marked stimulation in the incorporation of [14C]bicarbonate in incubations with added ammonium ions. The incorporation into total uridine nucleotides (sigma UMP) was increased twofold in the presence of 5 mM ammonium ion, and approximately eightyfold into orotic acid. There was a parallel increase in labelling of carbamoylaspartic acid from undetectable to a level similar to that of orotic acid. The specific activity of urea formed during the incubations did not change during incubations or in the presence of ammonium ions confirming that the change in labelling of pyrimidine was not due to a change in the specific activity of precursor. Despite the stimulation in incorporation of label into pyrimidines there was no increase in the hepatocyte content of sigma UMP, which was 11.5 mumol/g dry weight, although the orotic acid content increased from 0.09 mumol/g dry weight in the absence of added ammonium ions (but in the presence of 2 mM L-glutamine) to 8.6 mumol/g dry weight with 5 mM ammonium ion. The stimulated incorporation of [14C]bicarbonate in the presence of 5 mM and 10 mM ammonium ion was shown to be due to a stimulated synthesis of carbamoyl phosphate, since greater than 80% of label in the uracil ring was present at position C-2. Incubation of hepatocytes in basal medium (Eagles) containing 2.5% foetal calf serum and 20 mM bicarbonate showed that there was a significant stimulation of pyrimidine synthesis with 1 mM ammonium ion. The stimulatory effect of ammonium ions on incorporation of bicarbonate into pyrimidines was almost completely reversed by 5 mM L-ornithine and was partially reversed by 1 mM L-ornithine. Evidence for a contribution of the urea cycle carbamoyl phosphate synthetase to pyrimidine synthesis is discussed.
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PMID:Effect of ammonium ion on pyrimidine synthesis de novo in isolated rat hepatocytes. 725 Jan 18

The role of glucocorticosteroid hormones in the developmental formation of carbamoyl-phosphate synthase, ornithine transcarbamoylase, arginase, glutamate dehydrogenase, tyrosine aminotransferase, glucose-6-phosphatase, hexokinase and glucokinase activities in rat liver was investigated. Steroid hormone producing glands were either inactivated by hypophysectomy (before birth) or removed by adrenalectomy and/or gonadectomy (after birth). These procedures strongly depressed corticosterone levels. Furthermore, they decreased enzyme activities when performed before birth or after the second postnatal week. However, adrenalectomy at 1 week of age was less effective: the developmental increases in carbamoyl-phosphate synthase, ornithine transcarbamoylase, arginase, tyrosine aminotransferase and glucose-6-phosphatase activity persisted despite the absence of increasing levels of circulating corticosterone.
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PMID:Multihormonal control of enzyme clusters in rat liver ontogenesis. I. Effects of adrenalectomy and gonadectomy. 727 92

Several models of the urea cycle with normal and deficient enzymes were simulated on a desk-top computer in order to test if a model fitting data of patients suffering from deficiencies of urea cycle enzymes could be obtained. Discrepancies were notable if only the four enzymes of the urea cycle were used. The effect of adding either n-acetylglutamate synthetase and CPS or the mitochondrial transport system of ornithine and its catabolism to the model were tested singly or combined. The use of the combined model with the rate constants adjusted to recent data of the human enzymes together with a transport system for N-acetylglutamate out of the mitochondrium improved the results. Computer simulations are thus a useful tool for testing concepts of the functioning of the urea cycle.
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PMID:Computer simulation of the urea cycle: trials for an appropriate model. 729 39

Carbamoyl phosphate synthetase from Escherichia coli catalyzes the synthesis of carbamoyl phosphate from bicarbonate, ammonia, and two molecules of MgATP. The enzyme is composed of two nonidentical subunits. The small subunit catalyzes the hydrolysis of glutamine to glutamate and ammonia. The large subunit catalyzes the formation of carbamoyl phosphate and has the binding sites for bicarbonate, ammonia, MgATP, and the allosteric ligands IMP, UMP, and ornithine. The allosteric ligands are believed to bind to the extreme C-terminal portion of the large subunit. Truncation mutants were constructed to investigate the allosteric binding domain. Stop codons were introduced at various locations along the carB gene in order to delete amino acids from the carboxy-terminal end of the large subunit. Removal of 14-119 amino acids from the carboxy-terminal end of the large subunit resulted in significant decreases in all of the enzymatic activities catalyzed by the enzyme. A 40-fold decrease in the glutamine-dependent ATPase activity was observed for the delta 14 truncation. Similar losses in activity were also observed for the delta 50, delta 65, delta 91, and delta 119 mutant proteins. However, formation of carbamoyl phosphate was detected even after the deletion of 119 amino acids from the carboxy-terminal end of the large subunit. No allosteric effects were observed for UMP with either the delta 91 or delta 119 truncation mutants, but alterations in the catalytic activity were observed in the presence of ornithine even after the removal of the last 119 amino acids from the large subunit of CPS. Six conserved amino acids within the allosteric domain were mutated.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Regulatory changes in the control of carbamoyl phosphate synthetase induced by truncation and mutagenesis of the allosteric binding domain. 757 87

The structural and functional domains of Escherichia coli carbamoyl phosphate synthetase (CPS) have been identified by limited proteolysis. Incubation of CPS with several proteases, including trypsin, chymotrypsin, subtilisin and endoproteinase Asp-N, under native conditions, causes a time-dependent loss of enzymatic activity and the generation of a common fragmentation pattern. Amino-terminal sequencing studies demonstrated that the initial cleavage event by trypsin occurred at the carboxy-terminal end of the large subunit. The ultimate fragments produced in most of the proteolysis studies, 35- and 45-kDa peptides, were derived from areas corresponding to the putative ATP binding regions. Substrate protection studies showed that the addition of ligands did not affect the final fragmentation pattern of the protein. However, ornithine and UMP were found to significantly reduce the rate of inactivation by inhibition of proteolytic cleavage. MgATP and IMP provided modest protection whereas bicarbonate and glutamine showed no overall effect on proteolysis. Limited proteolysis by endoproteinase Asp-N resulted in the production of a fragment (or multiple fragments) which contained enzymatic activity but had lost all regulation by the allosteric ligands, UMP and ornithine. The small subunit has been shown to be protected from proteolysis by the large subunit. Proteolysis of the isolated small subunit resulted in the generation of a stable 31-kDa species which contained 10% of the original glutaminase activity. These studies demonstrate that a portion of the C-terminal end of the large subunit can be excised without entirely destroying the ability of CPS to catalyze the formation of carbamoyl phosphate.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Mapping the structural domains of E. coli carbamoyl phosphate synthetase using limited proteolysis. 764 1

The synthesis of citrulline from glutamine was quantified in enterocytes from pre-weaning (14-21 days old) and post-weaning (29-58 days old) pigs. The cells were incubated at 37 degrees C for 30 min in Krebs-Henseleit bicarbonate buffer (pH 7.4) containing 0, 0.5, 2 and 5 mM glutamine. Oxygen consumption was linear during the 30 min incubation period. The rates of citrulline synthesis were low or negligible in enterocytes from 14-21-day-old pigs, but increased 10-20-fold in the cells from 29-58-day-old pigs. This marked elevation of citrulline synthesis coincided with an increase in the activity of pyrroline-5-carboxylate synthase with the animal's post-weaning growth. In contrast, decreases in the activities of phosphate-dependent glutaminase, ornithine aminotransferase, ornithine carbamoyltransferase and carbamoyl-phosphate synthase were observed as the age of the pigs increased. The concentrations of carbamoyl phosphate in enterocytes from pre-weaning pigs were higher than, or similar to, those in the cells from post-weaning pigs. It is possible that the low rate of citrulline synthesis from glutamine in enterocytes from pre-weaning pigs was due to a limited availability of ornithine, rather than that of carbamoyl phosphate. We suggest that this limited availability of ornithine in pre-weaning-pig enterocytes results from (i) the low rate of pyrroline-5-carboxylate synthesis from glutamate, due to the low activity of pyrroline-5-carboxylate synthase, and (ii) the competitive conversion of pyrroline-5-carboxylate into proline. Our present findings on the developmental aspect of citrulline synthesis in pig enterocytes may offer a biochemical mechanism for the previous observations that arginine is a nutritionally essential amino acid for suckling piglets, but not for adult pigs.
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PMID:Synthesis of citrulline from glutamine in pig enterocytes. 816 28

Three conserved histidine residues, His-243, His-781, and His-788, located within the large subunit of carbamoyl phosphate synthetase from Escherichia coli were identified by sequence identity comparisons. These three histidine residues were individually mutated to asparagine residues. The H243N mutant enzyme was found to be critical for carbamoyl phosphate synthesis as the mutant protein was unable to synthesize carbamoyl phosphate at a significant rate (< 1/1500). By analysis of the effects of this mutation on the partial reactions catalyzed by CPS, it was determined that this mutation blocked the formation of the carbamate intermediate from carboxyphosphate and ammonia. The H781N mutant enzyme had an order of magnitude reduction for both the rate of carbamoyl phosphate formation and ATP synthesis which is consistent with the proposal that the carboxyl-terminal half of the large subunit is primarily involved in the phosphorylation of the putative carbamate intermediate. This mutation also reduced the effects of the allosteric activator ornithine on the Km parameters for ATP in the overall biosynthetic reaction and ADP in the ATP synthesis reaction. The H788N mutant enzyme is a functional protein which maintains the ability to synthesize carbamoyl phosphate at a rate comparable to that of the wild-type enzyme. The effects of this mutation are 10-fold reductions of the ATP synthetase and the bicarbonate-dependent ATPase activities with substantial increases in the Km values for ATP in the full biosynthetic reaction and for ADP in the ATP synthesis reaction.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Differential roles for three conserved histidine residues within the large subunit of carbamoyl phosphate synthetase. 841 43

Differential scanning calorimetry of Escherichia coli carbamoyl-phosphate synthetase and its isolated large and small subunits reveals in each case an irreversible, kinetically controlled transition, at a temperature 14 degrees C higher for the holoenzyme than for the subunits, indicating dramatic stabilization of the subunits in the heterodimer. The deletion of the COOH-terminal 171 (mutant CarB'2373) or 385 (mutant CarB2177) residues of the large subunit results in more asymmetric transitions at a temperature 7 degrees C lower than for the wild type. The allosteric effectors IMP, UMP, and ornithine induce small reversible transitions at low temperature in the endotherm for the wild-type enzyme, but not for CarB'2373, as expected if the effectors bind in the 171-residue, COOH-terminal region. In contrast, two ligands that bind outside the deleted region, Ap5A (a ligand of both ATP sites) and glycine (an analog of glutamine) decrease and increase, respectively, the stability of the two mutants and of the wild type. The stabilization by glycine requires that the subunits are associated. The results support the implication of the 20-kDa COOH-terminal domain of the large subunit in the allosteric modulation by all the effectors and are consistent with the folding of the large subunit as a pseudohomodimer of its two homologous halves.
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PMID:The influence of effectors and subunit interactions on Escherichia coli carbamoyl-phosphate synthetase studied by differential scanning calorimetry. 850 90


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