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)

Different fixation media have been compared in order to find one that preserves the histological structure of rat liver and allows unambiguous immunohistochemical detection of carbamoyl-phosphate synthetase (ammonia). Fixation of rat liver in a mixture of methanol, acetone, and water yields the most intense immunohistochemical staining. Using a specific antiserum raised against rat liver carbamoyl-phosphate synthetase, less than 1% of the enzyme protein is extractable after this fixation procedure, and the histological structure is similar to that after fixation in Bouin's fixative. Specific immunohistochemical staining is localized exclusively in the cytoplasm of the parenchymal cells; its granular distribution is in accordance with the mitochondrial localization of carbamoyl-phosphate synthetase. Immunohistochemical staining shows a heterogeneous distribution within the liver acinus. Staining is most intense around the portal venules, decreases slowly toward the hepatic venules and is, after an abrupt decrease, virtually absent in a limited area surrounding these venules. The possible significance of the heterogeneous distribution of carbamoyl-phosphate synthetase for ammonia metabolism is discussed.
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PMID:Immunohistochemical localization of carbamoyl-phosphate synthetase (ammonia) in adult rat liver; evidence for a heterogeneous distribution. 637 12

The pyrimidine-3 gene of Neurospora crassa codes for a bifunctional enzyme catalysing the first two steps of the pyrimidine biosynthetic pathway. Difficulties have been experienced in purification due to the lability of the enzyme. The enzyme loses carbamoyl-phosphate synthetase (carbon-dioxide: ammonia ligase (ADP-forming, carbamate-phosphorylating), EC 6.3.4.16) activity and undergoes a change in apparent molecular weight from the native 650,000 to 100,000 of the only detectable fragment. Attempts have been made therefore to stabilize the enzyme so as to minimise these effects. Elastinal, a protease inhibitor, reduces the effects, as do certain ultraviolet-sensitive mutant strains which lack a minor protease. The nature of the loss of carbamoyl-phosphate synthetase suggests an instability in the tertiary structure of the enzyme which can be reduced by the use of glycerol. Glycerol also exhibits a protease-inhibiting effect in this system. Although a range of protease inhibtors and use of uvs mutants can reduce the rate of decay of carbamoyl-phosphate synthetase activity, only glycerol can stabilize the native molecular weight. Our results support the hypothesis that the loss of carbamoyl-phosphate synthetase activity and change in molecular weight of the enzyme is a three-step sequence of proteolysis, conformational shift and cleavage of a further non-covalent bond.
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PMID:The involvement of proteolysis in conformational stability of the carbamoyl-phosphate synthetase/aspartate carbamoyltransferase enzyme of Neurospora crassa. 645 44

Carbamyl phosphate synthetase-I (CPS-I) catalyzes the first reaction required for the conversion of ammonia to urea through the urea cycle. Severe CPS-I deficiency causes marked hyperammonemia with encephalopathy in infancy and usually results in death within the first few months of life. We describe a 33-year-old woman whose CPS-I activity is less than 5% of normal. She has had mild, intermittent symptoms throughout life but has never experienced severe encephalopathy. Although mildly retarded, she has no major neurological deficits. Therapy with a low-protein diet, lactulose, and sodium benzoate has prevented recurrence of hyperammonemia and symptoms. Cranial computed tomographic scans demonstrate prominent lucency of cerebral white matter, and cerebral evoked potential recordings indicate slowed central conduction. These findings suggest that the metabolic disturbances in this patient may have adversely affected central myelin formation or maintenance. This woman represents, to our knowledge, the oldest reported patient with CPS-I deficiency, and the case illustrates the need to consider urea cycle disorders in the differential diagnosis of intermittent neurological symptoms regardless of the patient's age.
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PMID:Clinical features of carbamyl phosphate synthetase-I deficiency in an adult. 646 66

To examine the beneficial effect of arginine on ammonia intoxication, rats were injected intraperitoneally with a single dose of NH4Cl (6.75 mmol/kg) with and without arginine (5.0 mmol/kg) or ornithine (5.0 mmol/kg). Arginine or ornithine reduced the blood ammonia nitrogen at 30 min after NH4Cl injection from 3,288 +/- 800 micrograms/dl (mean +/- SE) to 538 +/- 90 and 575 +/- 34 micrograms/dl, respectively. In rats administered this dose of NH4Cl, arginine or ornithine did not increase further the hepatic carbamoyl-phosphate synthetase (EC 6.3.4.16) activation by N-acetylglutamate beyond the effect of NH4Cl. However, arginine or ornithine did increase the hepatic citrulline and urea content as well as the plasma urea concentration in these NH4Cl-injected rats. In rats injected with four doses of NH4Cl (2.5 mmol/kg), arginine or ornithine pretreatment increased the urea excretion and normalized the orotic acid excretion. These results indicate that arginine mitigates ammonia intoxication in the rat by increasing ornithine carbamoyltransferase activity through increased ornithine availability and not via activation of N-acetylglutamate synthetase. By increasing ornithine carbamoyltransferase activity, ornithine enhances the conversion of ammonia to citrulline and urea.
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PMID:Mechanism of arginine protection against ammonia intoxication in the rat. 647 19

The enzymes and intermediate metabolite of pyrimidine biosynthesis and ammonia metabolism were studied during perinatal period in rats. The activity of carbamyl phosphate synthetase I(CPS I) in fetal rat liver was low up to the 19th day of gestation, but a rapid increase was observed on the 20th day of gestation. The activity of CPS I in adult liver was about three times as high as that on the 17th day of gestation in fetal rat liver. The activities of carbamyl phosphate synthetase II(CPS II) and aspartate transcarbamylase (ATC) in fetal rat liver were much higher than those in adult liver, but a rapid decrease was observed from the 17th day of gestation up to birth. The activities of CPS II and ATC in adult liver were about 5--10% of those on the 17th day of gestation. The change in orotate content during the perinatal period in fetal rat liver was parallel to changes in the activities of CPS I and ATC, and a rapid decrease in orotate content in the last gestational stage was related to a rapid decrease in CPS and ATC activities. These results indicate that the activities of CPS I and CPS II change from the end of the gestational stage up to birth, and the proposed metabolic regulation of fetal growth and developments of considerable interest.
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PMID:[Ammonia metabolism during perinatal period--ontogenesis of enzymes in pyrimidine biosynthesis and urea cycle system]. 652 Apr 73

The glutamine- and N-acetyl-L-glutamate-dependent carbamoyl phosphate synthetase III present in liver of largemouth bass (Micropterus salmoides) has been highly purified. The properties of the enzyme are generally similar to the properties of the carbamoyl phosphate synthetase III from spiny dogfish (Squalus acanthias) previously described (Anderson, P. M. (1981) J. Biol. Chem. 256, 12228-12238). However, the bass enzyme is not subject to self-association, and the effects of urea and, particularly, trimethylamine-N-oxide, on catalytic activity are considerably reduced. Ammonia can substitute for glutamine as the nitrogen-donating substrate, but the maximum rate is lower. Carbamoyl phosphate synthetase III, like other carbamoyl phosphate synthetases, catalyzes two partial reactions, ATP synthesis from carbamoyl phosphate and ADP, and bicarbonate-dependent hydrolysis of ATP; both reactions are greatly stimulated by the presence of N-acetyl-L-glutamate. Carbamoyl phosphate synthetase III gave no detectable immunological cross-reaction with antibody to the ammonia- and N-acetyl-L-glutamate-dependent carbamoyl phosphate synthetase from rat liver mitochondria. The apparent Km value for N-acetyl-L-glutamate decreases significantly as the concentration of L-glutamine increases in the glutamine-dependent reaction, and vice versa. This effect is glutamine-specific. The apparent Km for N-acetyl-L-glutamate in the ammonia-dependent reaction is not affected by changes in ammonia concentration and the apparent Km for ammonia (8 mM) is also not affected by changes in N-acetyl-L-glutamate concentration. Studies involving inhibition of carbamoyl phosphate synthetase III by the glutamine analogs acivicin (L-(alpha S,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid), DON (6-diazo-5-oxo-L-norleucine), and chloroketone (L-2-amino-4-oxo-5-chloropentanoic acid), provided additional evidence for significant interaction between the L-glutamine- and N-acetyl-L-glutamate-binding sites. Glutamine-dependent but not ammonia-dependent activity is inhibited by preincubating the enzyme with these analogs. This inhibition requires the presence of both MgATP and N-acetyl-L-glutamate, and is prevented by the additional presence of L-glutamine. Inhibition of the glutamine-dependent reaction by DON or chloroketone is accompanied by a decrease in the apparent Km for N-acetyl-L-glutamate in the ammonia-dependent reaction from 0.3 mM to a value which is nearly the same as that observed in the glutamine-dependent reaction when glutamine is saturating (0.015 mM).
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PMID:Glutamine- and N-acetyl-L-glutamate-dependent carbamoyl phosphate synthetase from Micropterus salmoides. Purification, properties, and inhibition by glutamine analogs. 660 5

Shortly after birth, a newborn girl developed anorexia, hypotonia, apneic attacks and seizures. After 61 h the child died in coma. Biochemically, a highly elevated blood ammonia level was found together with an increased plasma level of the amino acids mainly involved in ammonia detoxication. Enzyme studies in post-mortem liver tissue material revealed a deficiency of carbamoyl-phosphate synthetase (0.9% of the mean value in controls) in combination with an intermediate activity of L-ornithine: 2-oxoglutarate aminotransferase (40% of the mean value in controls).
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PMID:A lethal neonatal variant of carbamoyl-phosphate synthetase deficiency in combination with an intermediate activity of L-ornithine: 2-oxoglutarate amino-transferase. 685 Dec 28

All six enzymes of the de novo biosynthetic pathway leading to the biosynthesis of UMP have been characterized in Toxoplasma gondii. The first three enzymes of the pathway, carbamyl phosphate synthetase-II (CPS-II), aspartate transcarbamylase (ATCase) and dihydroorotase (DHOase) could be consistently separated by sucrose gradient centrifugation. Their molecular weights were estimated to be approximately 540 000, 140 000 and 70 000, respectively. The last two enzymes, orotate phosphoribosyltransferase (OPRTase) and orotidylate decarboxylase (ODCase), cosedimented at the same position, corresponding also to a molecular weight of approximately 70 000. The fourth enzyme, dihydroorotate dehydrogenase (DHO-DHase), was associated with the particulate fraction. Apparent Km values for the respective enzymes were: CPS-II, MgATP2- (19.7 1.2 mM), L-glutamine (12.0 +/- 1.7 microM), ammonia (15.5 +/- 2.7 mM); ATCase, carbamyl phosphate (26.2 +/- 3.5 microM), L-aspartate (17.6 +/- 8.5 mM); DHOase (reverse direction) dihydroorotate (1.6 +/- 0.08 microM); ODCase, orotidine 5'-monophosphate (0.41 +/- 0.04 microM). MgUTP2- was found to act as an inhibitor of CPS-II, with an apparent Ki of 0.41 mM. However, 5-phospho-alpha-D-ribosyl-1-diphosphate, dimethyl sulphoxide and glycerol had no effect on the Km value for MgATP2-. The effect of some inhibitors, including pyrimidine and purine nucleotides and analogs and respiratory chain inhibitors, was also determined for the enzymes of the pathway.
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PMID:Enzymes of the de novo pyrimidine biosynthetic pathway in Toxoplasma gondii. 685 12

Some properties of carbamoyl-phosphate synthetase (ammonia) were studied in rat-liver mitochondria made selectively permeable by pretreatment with toluene. The Michaelis constants for NH3, MgATP and HCO-3 were 0.7, 1.2 and 2 mM respectively. N-Acetylglutamate activated the enzyme with a Ka of about 0.1 mM. At saturating concentrations of substrates and effectors the enzyme was inhibited by 50% by carbamoyl phosphate at a concentration of 13 mM. Binding of N-acetylglutamate to carbamoyl-phosphate synthetase required the presence of both free Mg2+ ions and MgATP, and was inhibited by Ca2+ ions and by N-carbamoylglutamate. The known activation of carbamoyl-phosphate synthetase by free Mg2+ is due to an increased affinity of the enzyme for N-acetylglutamate. Binding of N-acetylglutamate to carbamoyl-phosphate synthetase was a slow process: at N-acetylglutamate concentrations below 0.5 mM maximal binding was not completed within 30 min. The rate of binding increased with increasing N-acetylglutamate concentrations. Dissociation of N-acetylglutamate from the enzyme was relatively fast, with a half-time of about 5 min. Under all conditions studied there was a close relationship between carbamoyl-phosphate synthetase activity and the amount of N-acetylglutamate bound to the enzyme. The data are discussed in relation to the control of carbamoyl-phosphate synthetase in the intact hepatocyte.
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PMID:Properties of carbamoyl-phosphate synthetase (ammonia) in rat-liver mitochondria made permeable with toluene. 688 64

The binding of N-acetyl-L-glutamate, the physiological allosteric activator, to rat liver carbamoyl-phosphate synthetase (ammonia) was studied by techniques of rate of dialysis and of ultracentrifugation in the Airfuge. There is one binding site for acetylglutamate per enzyme monomer (Mr 165 000). K+, Mg2+ (free) and ATP were required to demonstrate binding. The concentrations of ATP required indicate that binding of ATPA (the ATP molecule that yields Pi) is needed. HCO-3 was not essential, but it enhanced binding of acetylglutamate. Glycerol also favored binding. Plots of Kd values versus the reciprocal of free Mg2+ and ATP concentrations are linear and indicate that ATPA, K+ and Mg2+ bind before acetylglutamate. In the presence of these ligands and HCO-3, ammonia increased drastically the Kd value for acetylglutamate, whereas in absence of HCO-3 ammonia had little effect. This suggests that acetylglutamate dissociates with the products and explains the higher Km for acetylglutamate in the synthetase (overall) reaction than in the ATPase (partial) reaction. In the absence of ATP acetylglutamate was bound with high affinity if ADP and carbamoyl phosphate were present. ADP or carbamoyl phosphate alone did not promote substantial binding. Binding of acetylglutamate at low concentration was slow; it was accelerated at higher concentrations of the activator. Exchange of bound acetylglutamate with acetylglutamate in solution was fast. A scheme proposed earlier for allosteric activation of the enzyme [Rubio, V., Britton, H. G. and Grisolia, S. (1983) Eur. J. Biochem. (in preparation)] is refined to incorporate the new information. Binding of ATPA, K+ and Mg2+ and formation of 'active CO2' (the central complex) are greatly favored by acetylglutamate.
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PMID:Binding of N-acetyl-L-glutamate to rat liver carbamoyl phosphate synthetase (ammonia). 688 68


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