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)

Acetylglutamate in HClO4 tissue extracts is first separated from glutamate by ion exchange. It is then deacylated with aminoacylase, and the resulting glutamate, after adsorption to and elution from an AG 50 column, is quantitated by a fast-HPLC method using o-phthaldialdehyde precolumn derivatization, separation in a C18 reverse-phase column, and fluorescence detection. A linear response is obtained up to 2 nmol, the detection limit is 5 pmol, and the method is suitable for assay in 1 mg liver tissue and thus for needle biopsies. When samples were analyzed by this procedure and by earlier procedures based upon detection of glutamate with glutamate dehydrogenase or upon activation of carbamoyl phosphate synthetase the results were similar. The method, which is highly specific, compares favorably in sensitivity, precision, and accuracy with all other published procedures. Using this assay, no acetylglutamate has been found in chicken liver and rat kidney.
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PMID:Determination of N-acetyl-L-glutamate using high-performance liquid chromatography. 399 35

Rats given a lethal dose (LD(99.9)) of ammonium acetate (10.8 mmol/kg of body weight) were protected to the extent of 85 and 76% when previously injected with N-carbamoyl glutamate or L-arginine, respectively, at a level of 4 mmol/kg of body weight. At a dose of 1 mmol/kg of body weight, L-arginine protected 24%, while N-carbamoyl-L-glutamate protected 61% of the animals. When a combination of N-carbamoyl-L-glutamate plus L-arginine (1 mmol each per kg of body weight) was injected, 100% of the rats were protected. The efficacy of N-carbamoyl-L-glutamate is related to its role as an activator of mitochondrial carbamoyl phosphate synthetase (EC 2.7.2.5) and its resistance to hydrolysis by tissue acylaminoacid acylase. N-Acetyl-L-glutamate, the naturally occurring and most effective activator of mitochondrial carbamoyl phosphate synthetase, was relatively ineffective in protection against lethal dose of ammonium acetate, because of its ready hydrolysis by acylaminoacid acylase. The findings reported provide a rational basis for the use of N-carbamoyl-L-glutamate plus L-arginine in the prevention and treatment of hyperammonemia in clinical conditions of liver disease and parental infusion of amino acids, and in feeding of urea supplements to ruminants.
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PMID:Ammonia intoxication in rats: protection by N-carbamoyl-L-glutamate plus L-arginine. 450 11

In the course of studies on glutamine-dependent carbamyl phosphate synthetase from Aerobacter aerogenes, we purified another protein which was found to be glutamate synthase (EC 2.6.1.53). The enzyme, obtained in apparently homogeneous form (monomer molecular weight about 227,000; s(20,omega) = 17.6 S), was found to be a typical glutamine amidotransferase in that it exhibits glutaminase activity and can utilize ammonia in place of glutamine as a nitrogen donor. The enzyme also catalyzes at low rates the oxidative deamination of glutamate in the presence of TPN, and it exhibits TPNH oxidase activity. The enzyme is similar to the glutamate synthase found in Escherichia coli in that it is an iron-sulfide flavoprotein. Treatment of the enzyme with sodium dodecyl sulfate or potassium thiocyanate dissociates it into nonidentical subunits exhibiting molecular weights of about 175,000 and 51,500. The glutamine-dependent activity of the enzyme is inhibited by L-2-amino-4-oxo-5-chloropentanoic acid, but this chloroketone analog of glutamine does not affect the ammonia-dependent glutamate synthase activity. Studies with [(14)C]chloroketone show that the reagent binds to the heavy subunit only. Inhibition by the chloroketone and its binding to the heavy subunit are markedly reduced in the presence of L-glutamine. Sedimentation velocity studies carried out in potassium thiocyanate indicate that iron-sulfide and flavin sites are also located on the heavy subunit. While these studies show that glutamate synthase resembles other glutamine amidotransferases in certain of its catalytic properties, the findings indicate that the light subunit of this enzyme, in contrast to that of several other glutamine amidotransferases, does not function to bind glutamine. It is of interest that the enzyme exhibits an unusually high affinity for ammonia as compared to a number of other glutamine amidotransferases. Glutamate synthase is inhibited (competitively with respect to glutamine) by low concentrations of methionine sulfone, methionine sulfoximine, and methionine sulfoxide.
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PMID:Glutamine-binding subunit of glutamate synthase and partial reactions catalyzed by this glutamine amidotransferase. 453 Oct 4

High levels of both glutamine synthetase and a unique L-glutamine- and N-acetyl-L-glutamate-dependent carbamoyl phosphate synthetase are present in the mitochondria in livers of marine urea-retaining elasmobranchs (Casey, C. A., and Anderson, P. M. (1982) J. Biol. Chem. 257, 8449-8453). On the basis of these observations it has been suggested that in these species carbamoyl phosphate and, consequently, one of the nitrogen atoms of citrulline and, ultimately, urea, are derived directly from glutamine rather than from ammonia as occurs in mammalian ureotelic species. The purpose of this study was to obtain evidence for this role of glutamine. Isolated hepatic mitochondria from Squalus acanthias incubated with ammonia plus glutamate, ornithine, bicarbonate, inorganic phosphate, and succinate as an energy source were found to synthesize citrulline at a rate comparable to the rate of urea synthesis observed in vivo. Citrulline synthesis proceeds at maximal rates even when the ammonia concentration is as low as 0.05 mM and is stoichiometric with the amount of ammonia initially present. Synthesis from ammonia does proceed in the absence of glutamate, but a much higher concentration of ammonia (congruent to 4 mM) is required to achieve a half-maximal rate. Glutamine can substitute for ammonia plus glutamate as the nitrogen-donating substrate for citrulline synthesis. Selective inhibition of the glutamine-dependent activity of the carbamoyl phosphate synthetase in the isolated mitochondria completely inhibits the ability of the mitochondria to synthesize citrulline from glutamine or from ammonia plus glutamate, whereas selective inhibition of glutamine synthetase inhibits citrulline synthesis from ammonia plus glutamate, but not from glutamine. These observations provide direct evidence that ammonia assimilation for citrulline synthesis (and, therefore, urea synthesis) in these species involves intermediate formation of glutamine.
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PMID:Glutamine-dependent synthesis of citrulline by isolated hepatic mitochondria from Squalus acanthias. 614 86

The pyr-3 gene of Neurospora crassa codes for the bifunctional enzyme pyrimidine-specific carbamoyl-phosphate synthetase/aspartate carbamoyltransferase (carbon dioxide: ammonia ligase (ADP-forming, carbamate-phosphorylating)/carbamoylphosphate: L-aspartate carbamoyltransferase), EC 6.3.4.16/EC 2.1.3.2). We describe the investigation of substrate- and product-binding sites of the enzyme by affinity chromatography, using the ligands aspartate, glutamate, and adenosine 5'-diphosphate, and investigate the channelling of carbamoyl phosphate, the product of the first function and substrate of the second, through the pathway. For this latter aspect of the investigation, two new enzyme assays were devised and described. The results of the competition studies on carbamoyl phosphate-binding are consistent with the existence of two different binding sites within the enzyme for this metabolic intermediate, one for it as the product of the first step and the other for it as the substrate of the second.
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PMID:Investigation of binding sites in the complex pyrimidine-specific carbamoyl-phosphate synthetase/aspartate carbamoyltransferase enzyme of Neurospora crassa. 621 40

High levels of glutamine- and N-acetyl-L-glutamate-dependent carbamoyl phosphate synthetase activity are present in liver extracts of marine species of fish that retain high levels of urea in their tissues for the purpose of osmoregulation. The function of the synthetase in these species appears to be related to urea synthesis.
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PMID:Glutamine- and N-acetylglutamate-dependent carbamoyl phosphate synthetase in elasmobranchs. 624 45

Reuber hepatoma H-35 was found to retain the activity of carbamoyl-phosphate synthetase I. The content of this enzyme in H-35 grown in Eagle's minimal essential medium was about half that in rat liver. The enzyme from H-35 was the same as that from rat liver in molecular weight estimated by SDS-polyacrylamide gel electrophoresis, specific enzyme activity, kinetic parameters for ATP and N-acetyl-L-glutamate, and immunological crossreactivity. The enzyme in H-35 was induced by dexamethasone (1.4-fold) but not by glucagon or dibutyryl cAMP. Incorporation of [35S] methionine into the enzyme indicated that the effect of dexamethasone was due to increased synthesis of the enzyme protein (2.1-fold). By labeling with [35S]methionine, the precursor and the mature forms of carbamoyl-phosphate synthetase I were observed in the post-mitochondrial and mitochondrial fractions, respectively. By chasing the labeled cells with unlabeled methionine and cycloheximide, it was observed that the rate of translocation of the precursor into mitochondria is not affected by dexamethasone.
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PMID:Induction of carbamoyl-phosphate synthetase I in Reuber hepatoma H-35 by dexamethasone. 630 26

Two paramagnetic probes, viz., Mn2+ and Cr3+-ATP, were used to map distances to various loci on carbamoyl-phosphate synthetase by using NMR measurements. The paramagnetic influence of Mn2+ on the 1H of L-glutamate and L-ornithine was measured at 200 and 360 MHz. On the basis of these data, a correlation time for the paramagnetic interaction was determined (2 X 10(-9) s) and used to compute distances. These were in the range 7-9 A. Distances were also calculated from Mn2+ to the 13C-5 atom of glutamate (8.6 A), to the monovalent cation site (approximately 8 A), and to the phosphorus atoms of ATP in the Co(NH3)4ATP complex. For studies of the monovalent cation site relaxation rates of 6Li+, 7Li+, and 15NH4+ were measured. With Cr3+ ATP as a paramagnetic substrate analogue, Cr3+ to 13C distances were measured with the substrates HCO3(-) and [5-13C]glutamate. These NMR data provide the first topographical map of the arrangement of substrates, metal ion activators, and allosteric modifiers on the Escherichia coli carbamoyl-phosphate synthetase dimer.
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PMID:A nuclear magnetic resonance study of the topography of binding sites of Escherichia coli carbamoyl-phosphate synthetase. 634 70

Valproate (0.5-5 mM) strongly inhibited urea synthesis in isolated rat hepatocytes incubated with 10 mM-alanine and 3 mM-ornithine. Valproate at the same concentrations markedly decreased concentrations of N-acetylglutamate, an essential activator of carbamoyl-phosphate synthetase I (EC 6.3.4.16), in parallel with the inhibition of urea synthesis by valproate. This compound also lowered the cellular concentration of acetyl-CoA, a substrate of N-acetylglutamate synthase (EC 2.3.1.1); glutamate, aspartate and citrulline were similarly decreased. Valproate in a dose up to 2 mM did not significantly affect the cellular concentration of ATP and had no direct effect on N-acetylglutamate synthesis, carbamoyl-phosphate synthetase I and ornithine transcarbamoylase (EC 2.1.3.3) activities.
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PMID:Inhibition of ureagenesis by valproate in rat hepatocytes. Role of N-acetylglutamate and acetyl-CoA. 641 45

Rat liver carbamoyl phosphate synthetase I is inactivated by elastase. Addition of ATP, Mg2+, K+ and N-acetyl-L-glutamate (the physiological allosteric activator) protects entirely, whereas acetylglutamate alone speeds inactivation. We have exploited these properties to investigate binding of these ligands. Acetylglutamate binds with low affinity (KD 0.25 mM) in the absence of other ligands, and with higher affinity (KD much less than 0.1 mM) when ATP, Mg2+ and K+ are present. The apparent KD for ATP in the presence of acetylglutamate is intermediate between the KD values for the two ATP binding sites present in the enzyme; thus, binding of ATP to both sites is involved in protecting the synthetase. The data also indicate binding of MgATP and Mg2+ in the absence of acetylglutamate. The results provide further evidence for conformational changes associated with allosteric activation of the enzyme.
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PMID:Inactivation of carbamoyl phosphate synthetase (ammonia) by elastase as a probe to investigate binding of the substrates. 655 79


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