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)

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

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

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

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

Independently folded structural domains of rat liver carbamoyl phosphate synthetase I have been identified by partial proteolytic cleavage under nondenaturing conditions. The pattern of fragments produced was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The NH2-terminal sequences of the fragments were determined by automated Edman degradation. Comparison of these fragment sequences with the sequence of the intact protein allowed alignment of the fragments. The hydrolysis of carbamoyl phosphate synthetase I (Mr 160,000) by either trypsin or elastase proceeded in two stages, with two alternative routes of degradation for elastase. The alignment of the final tryptic fragments from the NH2 terminus to the COOH terminus was: Mr 87,000 fragment-Mr 62,000 fragment-group of small peptides. The alignment of the final elastase fragments was: Mr 37,000 fragment-Mr 108,000 fragment-group of small peptides. The rates of cleavage were affected by the presence of the substrate ATP or the positive allosteric effector N-acetylglutamate; the preferred route of elastase cleavage was also affected. In addition to providing a map of the carbamoyl phosphate synthetase I domains and preliminary information on the interaction of substrates with these domains, the present studies provide further support for the proposal that domains serve as units of protein evolution since the 37-kDa fragment encompasses the region of the rat liver synthetase that is homologous to the 40-kDa subunit of the Escherichia coli synthetase.
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PMID:Domain structure of rat liver carbamoyl phosphate synthetase I. 349 Oct 68

In Crithidia fasciculata, carbamoyl phosphate synthetase II, which catalyses the first step of de novo pyrimidine biosynthesis, was separated from aspartate carbamoyltransferase by ammonium sulfate fractionation. The antitumor drug acivicin competitively inhibited the synthetase II activity with respect to L-glutamine, yielding an apparent Ki of 2 microM. In the absence of L-glutamine, acivicin resulted in a selective, time-dependent inactivation of L-glutamine-dependent activity of the enzyme, with an inactivation constant (Kinact) of 100 microM and a minimum inactivation half-time (T) of 0.2 min. L-Glutamine protected the enzyme from inactivation. These results are consistent with a postulate that acivicin is an active site-directed affinity analogue of L-glutamine, achieving irreversible inactivation. The inactivated enzyme retained ammonia-dependent activity. Acivicin stimulated the ammonia-dependent activity by increasing the Vmax value of the enzyme; apparent Km values for ammonia and MgATP were not affected. Differential action of acivicin on the Crithidia and mammalian synthetase II is discussed.
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PMID:Inactivation of Crithidia fasciculata carbamoyl phosphate synthetase II by the antitumor drug acivicin. 357 57

A high specific activity of carbamoyl-phosphate synthetase II (glutamine-hydrolyzing; EC 6.3.5.5) was demonstrated in extract of the cultured Crithidia fasciculata. The enzyme was separated from aspartate carbamoyltransferase by ammonium sulfate fractionation. Apparent Km for the synthetase for L-glutamine, NH4+, MgATP or bicarbonate was 0.27, 26, 1.7 or 1.7 mM at 2.0% dimethyl sulfoxide plus 0.3% glycerol. 8.6% dimethyl sulfoxide plus 1.4% glycerol decreased Km for L-glutamine to 0.10 mM, while Km for MgATP was unaffected. The higher solvent concentrations made Vmax markedly reduced, yielding the inhibition of the activity. These properties are unique to the Crithidia synthetase, compared with the mammalian enzyme.
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PMID:Kinetic properties of carbamoyl-phosphate synthetase II (glutamine-hydrolyzing) in the parasitic protozoan Crithidia fasciculata and separation of the enzyme from aspartate carbamoyltransferase. 360 29

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

In rat livers and hepatomas, carbamoyl phosphate synthetase (glutamine-hydrolyzing) (EC 6.3.5.5) (synthetase II), the rate-limiting enzyme of de novo pyrimidine nucleotide biosynthesis, was separated from carbamoyl phosphate synthetase (ammonia) (EC 6.3.4.16) (synthetase I) ammonium sulfate and hydroxylapatite fractionations and gel filtration on Sephadex G-25. Both liver and hepatoma 3924A synthetase II activities were subject to feedback inhibition by UTP and to stimulation by 5-phosphoribosyl 1-pyrophosphate. UTP (0.5 mM) enhanced the apparent Km for MgATP from 2.3 to 7.6 mM, whereas 0.1 mM 5-phosphoribosyl 1-pyrophosphate reduced it to 0.5 mM. At 2 mM MgATP, 3 or 7 microM 5-phosphoribosyl 1-pyrophosphate yielded half-maximal activation (Ka) in the absence or presence of 0.5 mM UTP; UTP altered the stimulation kinetics from hyperbolic to sigmoidal. In the rat, synthetase II activities were highest in thymus, testis and spleen. In differentiating and regenerating rat livers, activities were 2.2- and 1.5-fold higher than in adult livers. In 17 hepatomas of different growth rates, synthetase II activity increased 1.3- to 9.5-fold over liver values; the rise correlated positively with tumor growth rates. Synthetase II activities also increased in a kidney tumor (5.0-fold) and in a sarcoma (18.1-fold) in the rat and in a human colon tumor (3.3-fold).
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PMID:Regulatory properties and behavior of activity of carbamoyl phosphate synthetase II (glutamine-hydrolyzing) in normal and proliferating tissues. 705 79


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