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)

The possibility of control of the activity of carbamoyl-phosphate synthase (ammonia) (EC 2.7.2.5) in rat-liver mitochondria by variation in the intramitochondrial free Mg2+ concentration has been investigated. Carbamoyl-phosphate synthase activity was measured by coupling the formation of carbamoylphosphate to the synthesis of citrulline in a reaction mixture containing ammonia, bicarbonate, a source of ATP, and ornithine. The synthesis of citrulline was inhibited by lowering the concentration of intramitochondrial free Mg2+. This could be achieved not only by depleting the mitochondria of Mg2+ (by adding the ionophore A23187), but also by increasing the intramitochondrial concentration of citrate. Under various conditions an inverse relationship between the rate of citrulline synthesis and the magnitude of the intramitochondrial concentration of citrate was observed. Inhibition of citrulline synthesis by intramitochondrial citrate could be partly reversed by addition of Mg2+ in the presence of A23187. Possible implications of the regulation of carbamoyl-phosphate synthase (ammonia) activity by intramitochondrial citrate for nitrogen metabolism in the liver are discussed.
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PMID:Relationship between intramitochondrial citrate and the activity of carbamoyl-phosphate synthase (ammonia). 2 2

1. The influence of ammonia and ornithine on the oxygen uptake and the formation of citrulline was investigated with isolated rat liver mitochondria. The experiments were performed in a cytosol-like saline medium at 38 degrees C. 2. Under these conditions an increase of the respiration rate by ammonia and ornithine was observed, but a small response to external ADP, only. The missing stimulation by ADP was due to a partial inhibition of the respiratory chain by traces of zinc (approximately 1 microM) present in the medium. This inhibition was only detected at low concentrations of mitochondria. 3. For activation of respiration by ammonia plus ornithine two different processes were responsible: (i) chelation of the inhibiting zinc by ornithine, which could be prevented by EDTA; (ii) ADP production in the matrix space during formation of carbamoyl phosphate, which could be prevented by oligomycin but not by carboxyatractyloside. 4. This stimulus of the carbamoyl phosphate formation and of the equivalent citrulline synthesis on the mitochondrial respiration ran to 12% of that increase caused by phosphorylation of external ADP. The maximum rate of citrulline formation was limited by the activity of carbamoyl phosphate synthetase. 5. Added ADP suppresses the production of citrulline probably by the exchange of extramitochondrial ADP versus intramitochondrial ATP. The data suggest a common adenine nucleotide pool delivering ATP to the adenine nucleotide translocase as well as to the carbamoyl phosphate synthetase.
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PMID:The stimulation of the mitochondrial respiration by citrulline synthesis. 11 92

Mutants resistant to 5-fluorouracil, 5-fluorouridine and 5-fluorodeoxyuridine have been selected in Aspergillus nidulans. Growth tests combined with genetic analysis showed that mutations conferring resistance to fluoropyrimidines could occur in at least seven genes. Three of these fulE, fulF and furA were concerned with either the uptake of pyrimidines or their conversion to uridine monophosphate. The other four genes did not affect these functions. Mutations in fulA probably confer resistance by lowering ornithine transcarbamoylase, thereby making the normally arginine-specific carbamoyl phosphate pool available for increased uracil synthesis. Mutations in fulD may make the arginine-specific carbamoyl phosphate synthetase insensitive to inhibition or repression by arginine, and so lead to increased carbamoyl phosphate pool sizes, and increased uracil synthesis. Both fulA and fulD mutants suppress pyrA mutants which lack the uracil-specific carbamoyl phosphate synthetase. Mutations in fulB and fulC do not suppress pyrA, and so may act more directly to increase uracil synthesis. The synthesis of aspartate carbamoyl transferase in fulB7 strains is not repressed by uracil. fulC mutants are closely linked to the pyrA, B, C, N region which codes for the first two enzymes of pyrimidine biosynthesis, and may result in these enzymes being less sensitive to inhibition by uracil.
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PMID:Pyrimidine biosynthesis in Aspergillus nidulans. Isolation and characterisation of mutants resistant to fluoropyrimidines. 12 29

The arginine-specific carbamoyl-phosphate synthase of yeast was stabilized sufficiently to allow partial purification of the enzyme (30- to 40-fold). The synthase (mol. wt 115000) comprised two unequal subunits: a heavy subunit (mol. wt 80000) capable of catalysing synthesis of carbamoyl phosphate with ammonia as a nitrogen donor and a light subunit conferring upon the holoenzyme the ability to utilize glutamine. The enzyme had unusually high affinity for ATP (Km = 0.2 mM) and atypical negative cooperativity for glutamine binding ([S]0.5 = 0.25 mM). Glutamine activity was not modulated by possible effectors such as arginine, ornithine or N-acetylglutamate. Thus, although the yeast arginine enzyme physically and functionally resembles the single enteric synthase, the systems differ substantially both in kinetic properties and in regulation of activity.
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PMID:Purification and properties of the arginine-specific carbamoyl-phosphate synthase from Saccharomyces cerevisiae. 20 52

Methods are described for the determination of the activity of urea cycle enzymes in human and rat tissues by chromatography and videodensitometry(CV-technique). With specific substrates carbamoyl-phosphate synthetase and ornithine carbamoyltransferase activities were determined as the amounts of citrulline formed. Argininosuccinate synthetase, argininosuccinate lyase and arginase activities were measured from the changes in ornithine concentration. For measuring the activity of five enzymes 5 to 10 mg wet weight of tissue was sufficient. The CV-technique could be conveniently applied for the investigation of enzyme content in samples from human biopsy.
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PMID:Determination of enzyme activity by chromatography and videodensitometry. II. Urea cycle enzymes in tissue homogenates. 23 8

The reaction of phenylglyoxal with two enzymes in which ATP plays a complex role has been studied. Both ovine brain glutamine synthetase and Escherichia coli carbamyl phosphate synthetase [carbamoyl-phosphate synthase (glutamine); ATP:carbamate phosphotransferase (dephosphorylating, amido-transferring); EC 2.7.2.9]were inactivated by phenylglyoxal. The specificity of this reagent for arginyl residues of the two proteins was confirmed by amino acid analysis. ATP, but not the other substrates, protected these enzymes against inactivation by phenylglyoxal. Carbamyl phosphate synthetase was also protected by IMP and ornithine, positive allosteric effectors that alter the enzymatic activity be increasing the affinity for ATP. UMP, a negative allosteric effector that decreases the affinity for ATP, did not protect against inactivation. Differential labeling experiments with [14C]phenylglyoxal showed that the number of arginyl residues protected by ATP corresponded quite well to the known number of ATP catalytic sites for each protein. These data indicate that arginyl residues at the active sites of glutamine synthetase and carbamyl phosphate synthetase are involved in the binding of ATP. This phenylglyoxal inactivation study also provided information about the mechanistic role of ATP in the two synthetases. The data obtained on glutamine synthetase support the theory that ATP is attached to the enzyme as a portion of the catalytic site, and that its presence is essential for the binding of glutamate and glutamine. The data obtained on carbamyl phosphate synthetase are consistent with the previous proposal that carbonyl phosphate is an intermediate in the ATP-dependent activation of bicarbonate by this enzyme. It is also of interest that, with both glutamine synthetase and carbamyl phosphate synthetase, only a small portion of the total arginyl population of these enzymes reacted with phenylglyoxal. A summary of previous studies on the modification of enzyme arginyl residues is presented.
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PMID:Functional arginyl residues as ATP binding sites of glutamine synthetase and carbamyl phosphate synthetase. 24 Oct 76

Previous studies using intact rat liver mitochondria have shown that the soluble matrix enzymes carbamoyl-phosphate synthase (ammonia) (CPS) and ornithine carbamoyltransferase (OCT) display some kinetic properties which would not be observed if they were homogeneously distributed in the matrix. In the present work we have extended these studies, using toluene-treated mitochondria which are fully permeable to substrates and inhibitors, yet retain 90% of their soluble enzymes. The results provide evidence of functional organization of CPS and OCT in situ. The major findings are as follows. (1) The apparent Km values of matrix OCT for carbamoyl phosphate and ornithine are respectively 8 and 2 times those measured for the soluble enzyme. delta-N-Phosphonacetyl-L-ornithine inhibits OCT in situ less than in solution, especially when carbamoyl phosphate is synthesized in the mitochondria rather than added to the medium. (2) During citrulline synthesis from endogenously generated carbamoyl phosphate, the concentration of the latter in permeabilized mitochondria is more than 10 times that in the medium, although the mitochondria are freely permeable to added molecules of this size. (3) Endogenously formed carbamoyl phosphate is used preferentially by OCT in situ; addition of a 200-fold excess of unlabelled carbamoyl phosphate has little effect on the conversion of labelled endogenously formed carbamoyl phosphate into citrulline by matrix OCT. (4) The synthesis de novo of carbamoyl phosphate from NH3, HCO3- and ATPMg is the same in the presence and absence of ornithine. (5) Studies with co-immobilized CPS and OCT gave results concordant with some of the above observations and with previous ones with intact mitochondria.
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PMID:Kinetic properties of carbamoyl-phosphate synthase (ammonia) and ornithine carbamoyltransferase in permeabilized mitochondria. 154 Jan 32

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)
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PMID: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. 186 Sep 1

The large subunit of Escherichia coli carbamoyl phosphate synthetase (a polypeptide of 117.7 kDa that consists of two homologous halves) is responsible for carbamoyl phosphate synthesis from NH3 and for the binding of the allosteric activators ornithine and IMP and of the inhibitor UMP. Elastase, trypsin, and chymotrypsin inactivate the enzyme and cleave the large subunit at a site approximately 15 kDa from the COOH terminus (demonstrated by NH2-terminal sequencing). UMP, IMP, and ornithine prevent this cleavage and the inactivation. Upon irradiation with ultraviolet light in the presence of [14C]UMP, the large subunit is labeled selectively and specifically. The labeling is inhibited by ornithine and IMP. Cleavage of the 15-kDa COOH-terminal region by prior treatment of the enzyme with trypsin prevents the labeling on subsequent irradiation with [14C]UMP. The [14C]UMP-labeled large subunit is resistant to proteolytic cleavage, but if it is treated with SDS the resistance is lost, indicating that UMP is cross-linked to its binding site and that the protection is due to conformational factors. In the presence of SDS, the labeled large subunit is cleaved by trypsin or by V8 staphylococcal protease at a site located 15 or 25 kDa, respectively, from the COOH terminus (shown by NH2-terminal sequencing), and only the 15- or 25-kDa fragments are labeled. Similarly, upon cleavage of the aspartyl-prolyl bonds of the [14C]UMP-labeled enzyme with 70% formic acid, labeling was found only in the 18.5-kDa fragment that contains the COOH terminus of the subunit. Thus, UMP binds to the COOH-terminal domain.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Domain structure of the large subunit of Escherichia coli carbamoyl phosphate synthetase. Location of the binding site for the allosteric inhibitor UMP in the COOH-terminal domain. 198 78

The rate of citrulline synthesis in mitochondria from OTC-deficient spf-ash mice (15% of the normal activity) was found to be the same as that in mitochondria from control mice. The amount of NAG in their mitochondria varied markedly according to whether they had received a high- or low-protein diet, and the rate of citrulline synthesis was found to be affected by the level of NAG. These results indicate that the CPS stage, not the OTC stage, is rate-limiting in the citrulline synthesis process. Kinetic studies on the effect of ornithine concentration on citrulline synthesis in mitochondria showed that the Km for ornithine was very low in the mitochondria from the mice given a low-protein diet. Kinetic studies on the effect of ornithine concentration on mouse OTC at various concentrations of carbamylphosphate showed that OTC has a ping-pong mechanism, i.e., that the Km for ornithine and Vmax decrease with the reduction in carbamylphosphate concentration. This may explain the low Km value observed in citrulline synthesis in the mitochondria. We conclude that in mitochondrial citrulline synthesis the rate of carbamylphosphate synthesis by CPS in the presence of NAG plays a key role in determining the rate of citrulline synthesis and ornithine dependency.
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PMID:Effect of ornithine concentration on citrulline synthesis in mouse liver mitochondria. 238 23


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