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)

Experiments were carried out to locate carbamoyl phosphate synthetase (CPS) in rat liver by direct immunoferritin labeling. By using Epon sections treated with sodium methoxide, homogenates or mitochondrial and mitoplast fractions, carbamoyl phosphate synthetase was found homogeneously distributed in the mitochondrial matrix. Immunoferritin was detected with high resolution which permits the identification of individual molecules. Measurements were made of the number of ferritin particles per square micron of mitochondrial surface, providing a novel and independent assessment of the carbamoyl phosphate synthetase concentration.
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PMID:Immunoferritin location of carbamoyl phosphate synthetase in rat liver. 9 72

Total RNA or poly(A)(+) RNA of rat liver was translated in a rabbit reticulocyte or wheat germ protein-synthesizing system and the carbamyl phosphate synthetase I [carbamoyl-phosphate synthetase (ammonia); carbon dioxide: ammonia ligase (ADP-forming, carbamate-phosphorylating), EC 6.3.4.16] synthesized was isolated by indirect immunoprecipitation by using antibody purified on enzyme-bound Sepharose and Staphylococcus aureus cells. The in vitro product moved on sodium dodecyl sulfate/polyacrylamide gels as a polypeptide that was about 5000 daltons larger than the subunit of the mature enzyme (160,000 daltons). The same polypeptide was also obtained by direct immunoprecipitation or by a double-antibody precipitation method. The mature enzyme competed effectively with the in vitro product for interaction with anti-carbamyl phosphate synthetase I antibody. Digestion of the in vitro product by S. aureus protease gave a pattern of peptide fragments similar to that of the mature enzyme. A mitochondrial membrane preparation from rat liver converted the in vitro product into a polypeptide that comigrated with the mature subunit on sodium dodecyl sulfate gel electrophoresis. Similar proteolytic activity was not detected in either a cytosol or a microsomal fraction of rat liver. These results indicate that the enzyme is synthesized as a larger precursor which is converted to the mature form of enzyme by posttranslational processing.
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PMID:Cell-free synthesis and processing of a putative precursor for mitochondrial carbamyl phosphate synthetase I of rat liver. 22 76

Ammoniacal silver solutions give striking impregnation of Alzheimer's disease (AD) lesions if sections are pretreated with copper sulfate and hydrogen peroxide. In contrast to most silver impregnation methods, no staining of normal neurites is obtained, and senile plaques (SPs), neurofibrillary tangles (NFTs), and neuropil threads (NTs) are strongly stained in black against a clear background. A sodium acetate wash interposed between the copper sulfate and hydrogen peroxide resulted in suppression of the staining of amyloid lesions. This variant of the basic procedure (CPS-II method), maintains the capacity of the latter (CPS-I method) to strongly impregnate NFTs and NTs. In addition, it clearly delineates the dystrophic neurites of SPs obscured by the strong argyrophilia of the amyloid deposits seen in CPS-I stains. NTs are strongly impregnated with both CPS-I and CPS-II methods and are unmasked from normal neurites, which remain unstained. The staining can be abolished by pretreatment with formic acid and erased with a brief wash in sulfochromic acid. Destained sections can be restained with either method or with immunoperoxidase procedures. CPS staining of previously immunostained tissues produces marked intensification of the diaminobenzidine reaction product. In AD brains, the immunostaining is markedly enhanced and selective when the silver procedure is preceded by formic acid treatment. The selectivity and high sensitivity of the procedure may be useful as a diagnostic tool and of value to study the biogenesis and natural evolution of the brain lesions of AD.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:The copper-peroxide-silver method: a highly sensitive procedure for the demonstration of Alzheimer's disease lesions and for signal intensification in immunocytochemistry. 149 51

Synthesis of glucose from lactate and generation of urea from ammonia were inhibited when sodium benzoate was added to suspensions of rat hepatocytes. Assays with isolated mitochondria suggested pyruvate carboxylase and the N-acetyl-L-glutamate (NAG)-dependent carbamoylphosphate synthetase (CPS-I) as potential sites of inhibition for both pathways, owing to a shared dependency on aspartate efflux from the mitochondria and its subsequent conversion to oxaloacetate in the cytosol. Assays with isolated hepatocytes indicated inhibition to be initiated by accumulation of benzoyl CoA with a resultant depletion of free CoA and acetyl CoA. Measurements of adenine nucleotides showed that benzoate metabolism did not sufficiently alter energy status to account for the observed inhibition. Consistent with these interpretations, acceleration of the conversion of benzoyl CoA to hippurate by the addition of glycine restored the levels of free CoA and acetyl CoA and the rates of gluconeogenesis and ureagenesis. Reduction of the levels of aspartate and glutamate, presumably by interference with the anapleurotic function of pyruvate carboxylase, most likely accounted for inhibition of gluconeogenesis by benzoate. Whether reduced flux through the urea cycle also contributed to inhibition of gluconeogenesis (by diminishing cytosolic conversion of aspartate to oxaloacetate) requires further study. Depression of glutamate and acetyl CoA to levels at or below the Km for NAG synthetase probably accounted for the observed inhibition of ureagenesis. Rates of urea production were observed to vary with changes in the levels of NAG, suggesting NAG-dependent CPS-I to be the primary site of inhibition of ureagenesis by benzoate.
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PMID:On the mechanism of inhibition of gluconeogenesis and ureagenesis by sodium benzoate. 167 73

Dihydroorotase (DHOase) catalyzes the reversible cyclization of N-carbamoyl-L-aspartate (L-CA) to L-5,6-dihydroorotate (L-DHO), which is the third enzyme in de novo pyrimidine biosynthesis. The enzyme was purified from two parasitic protozoa, Crithidia fasciculata (about 16,000-fold) and Plasmodium berghei (about 790-fold). The C. fasciculata enzyme had a native molecular weight (Mr) of 42,000 +/- 5000, determined by gel filtration chromatography, and showed a single detectable protein band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with Mr 44,000 +/- 3000. The DHOase from P. berghei had a native molecular weight of 40,000 +/- 4000 and a subunit molecular weight on SDS-PAGE of 38,000 +/- 3000. The DHOase from both parasites, in contrast to the mammalian enzyme which resides on a trifunctional protein of the first two enzymes of the pathway, carbamoyl-phosphate synthase and aspartate transcarbamylase, is monomeric and has no oligomeric structure as studied by chemical cross-linking with dimethyl suberimidate. The rate of cyclization of L-CA by the C. fasciculata enzyme was relatively high at acidic pH, decreasing to a very low rate at alkaline pH. In contrast, the rate of ring cleavage of L-DHO was very low at acidic pH and increased to a higher rate at alkaline pH. These pH-activity profiles gave an intersection at pH 6.6. The Km and kcat for L-CA were 0.846 +/- 0.017 mM and 39.2 +/- 6.4 min-1, respectively; for L-DHO, they were 25.85 +/- 2.67 microM and 258.6 +/- 28.5 min-1.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Pyrimidine biosynthesis in parasitic protozoa: purification of a monofunctional dihydroorotase from Plasmodium berghei and Crithidia fasciculata. 197 82

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.
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PMID:A case of carbamylphosphate synthetase-I deficiency associated with secondary carnitine deficiency--L-carnitine treatment of CPS-I deficiency. 230 75

Benzoylcarnitine was identified in the urine of a patient with a carbamoyl-phosphate synthase I deficiency for whom sodium benzoate and L-carnitine had been used to treat hyperammonemia. This is a newly identified metabolite of benzoate. Its excretion in the urine was increased day by day at the administration of both sodium benzoate and L-carnitine from 0.10 to 2.25 mmol/g creatinine. Since there is the possibility of a secondary carnitine deficiency and an increase of benzoyl toxicity after long-term therapy with benzoate supplementation and protein restriction, it is important to monitor the urinary excretion of benzoylcarnitine.
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PMID:Identification of benzoylcarnitine in the urine of a patient of hyperammonemia. 260 32

Dihydroorotase (DHOase) catalyzes the third step in eukaryotic de novo pyrimidine biosynthesis. In mammalian cells, this enzyme activity is carried by a large chimeric protein, CAD, that also catalyzes the first two steps in the pathway: glutamine-dependent carbamyl phosphate synthetase (CPSase) and aspartate transcarbamylase (ATCase). Controlled elastase cleavage of CAD released a 44,000 +/- 2,000-dalton proteolytic fragment which catalyzed only the dihydroorotase reaction. We have devised a rapid and simple method for the isolation of the DHO domain from elastase digests. The domain, which was obtained in 36% yield, was found to be homogeneous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing. The domain was also characterized by amino acid analysis and analytical high pressure liquid chromatography peptide mapping. The amino terminus of both the DHO domain and intact CAD was blocked suggesting that this domain is located at the extreme amino terminus of the CAD polypeptide, a result consistent with the suspected juxtaposition of domains as DHO-CPS-ATC. The isoelectric point of the DHO domain was 5.1, while that of the ATC domain was 9.4, so that the ends of the CAD polypeptide are oppositely charged at physiological pH. Immunoblotting with DHO domain-specific antibodies showed that a 47-kDa species was generated in the early stages of controlled proteolysis of CAD. Thus there are two elastase cleavage sites within a 3-kDa connecting region that links the DHO and CPS domains. The domain was shown by atomic absorption spectrophotometry and by isolating a 65Zn-containing DHO domain from mammalian cells grown in the presence of the radionuclide to contain 1 g eq of tightly bound zinc in each polypeptide chain. Zinc was not found in any other CAD domain. Chelating agents inhibit dihydroorotase activity of the isolated domain supporting the conclusion, based on studies of intact CAD by others, that zinc participates in catalysis. At moderate protein concentrations the DHO domain was a 88,000 dimer with a Stokes radius of 37.6 A, a S20,w = 5.1 X 10(-13) s, a diffusion coefficient of 3.17 X 10(-7) cm2 s-1, and a frictional ratio of 1.26. On dilution the dimer dissociated and was in rapid concentration-dependent equilibrium with a 43,500 monomer. The hydrodynamic parameters of the monomer have also been estimated (Stokes radius of 29.8 A, D20,w = 4.11 X 10(-7) cm2 s-1, and f/f0 1.21).(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:The dihydroorotase domain of the multifunctional protein CAD. Subunit structure, zinc content, and kinetics. 287 Oct 22

An indirect, competitive enzyme-linked immunosorbent assay for the quantitation of carbamoyl-phosphate synthetase I (ammonia) in rat liver has been developed. Homogenization of the liver in 1% sodium deoxycholate is used for complete solubilization of the enzyme. The detergent does not interfere with the method if diluted to a concentration of 0.01% or lower. The assay is applied to determine the amount of enzyme in control rats and in rats fed "cafeteria" or high-protein diets. Changes in the amount of carbamoyl-phosphate synthetase I (ammonia) paralleled changes in enzymatic activity.
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PMID:An enzyme immunoassay for the quantitation of rat liver carbamoyl-phosphate synthetase I. 307 Nov 80

A carbamoyl-phosphate synthase has been purified from mycelia of Phycomyces blakesleeanus NRRL 1555 (-). The molecular weight of the enzyme was estimated to be 188,000 by gel filtration. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate showed that the enzyme consists of two unequal subunits with molecular weights of 130,000 and 55,000. The purified enzyme has been shown to be highly unstable. The carbamoyl-phosphate synthase from Phycomyces uses ammonia and not L-glutamine as a primary N donor and does not require activation by N-acetyl-L-glutamate, but it does require free Mg2+ for maximal activity. Kinetic studies showed a hyperbolic behavior with respect to ammonia (Km 6.34 mM), bicarbonate (Km 10.5 mM) and ATP.2 Mg2+ (Km 0.93 mM). The optimum pH of the enzyme activity was 7.4-7.8. The Phycomyces carbamoyl-phosphate synthase showed a transition temperature at 38.5 degrees C. It was completely indifferent to ornithine, cysteine, glycine, IMP, dithiothreitol, glycerol, UMP, UDP and UTP. The enzyme was inhibited by reaction with 5 mM N-ethylmaleimide.
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PMID:Carbamoyl-phosphate synthase in Phycomyces blakesleeanus. 339 22


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