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)

1. At the lowered concentrations of 0.5 mM ATP and 1.5 mM MgCl2, 2.0 mM UTP, UDP and UMP inhibited the activity of Crithidia fasciculata carbamoyl-phosphate synthetase II by about 65, 80 and 40% respectively. 2. The result suggests that feedback inhibition of the activity by uridine nucleotides is a mechanism of regulation of the de novo pyrimidine biosynthetic pathway in C. fasciculata. 3. ADP, AMP and CDP inhibited the activity (about 70, 40 and 40%). 4. Excess Mg2+ at around 1 mM, relative to the ATP concentration, was required for the maximum activity. 5. 5-Phosphoribosyl 1-pyrophosphate had no significant effect on the activity under various conditions examined.
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PMID:Regulatory properties of carbamoyl-phosphate synthetase II from the parasitic protozoan Crithidia fasciculata. 244 85

CAD codes for a trifunctional protein involved in the catalysis of the first three enzymatic activities in the de novo pyrimidine biosynthetic pathway, namely, carbamoyl-phosphate synthetase II (EC 6.3.5.5), aspartate transcarbamylase (EC 2.1.3.2), and dihydroorotase (EC 3.5.2.3). CAD regulation was studied in the human promyelocyte leukemic line HL-60 as it differentiated into monocytic or granulocytic lineages after induction by 12-O-tetradecanoylphorbol-13-acetate or trans-retinoic acid and dibutyryl cyclic AMP, respectively. Within 12 h of induction of HL-60 cells with either inducer, total cellular levels of CAD RNA essentially disappeared. On the other hand, no apparent decreases in beta-actin RNA levels were seen even 48 h after HL-60 cells were induced, as compared with untreated cells. With nuclear runoff assays, it was clearly shown that the inactivation of CAD gene expression during the induction of HL-60 cells with either inducer was at the transcriptional level. The nuclear runoff experiments also demonstrated that the CAD gene expression was shut down in less than 4 h after induction, well before morphological changes were observed in these cells. At the enzymatic level, the activity of aspartate transcarbamylase, one of the three enzymes encoded by the CAD gene, decreased by about half within 24 h of induction, suggesting a CAD protein half-life of 24 h in differentiating HL-60 cells. Nevertheless, this means that significant levels of aspartate transcarbamylase activity remained even after the cells have stopped proliferating. From the RNA data, it is clear that CAD gene expression is rapidly turned off as promyelocytes begin to terminally differentiate into macrophages and granulocytes. We suspect that the inactivation of the CAD gene in induced HL-60 cells is a consequence of the differentiating cells leaving the cell cycle and becoming nonproliferating.
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PMID:Transcriptional regulation of the human CAD gene during myeloid differentiation. 288 43

We have studied the regulation of expression of the carbamoyl-phosphate synthetase II-aspartate transcarbamylase-dihydroorotase gene in F9 teratocarcinoma cells during their differentiation into parietal endoderm cells by induction with a combination of retinoic acid and dibutyryl cyclic AMP. Steady-state levels of CAD mRNA decreased by 7-fold in F9 cells following 120 h of retinoic acid and dibutyryl cyclic AMP induction as compared to levels in uninduced cells. Conversely, no apparent changes were found in the steady-state levels of beta-actin mRNA between induced and uninduced cells. Despite a 7-fold decrease in the steady-state levels of CAD mRNA, its rate of transcription remained the same between induced and uninduced cells, indicating a role for posttranscriptional mechanisms for its down regulation during retinoic acid- and dibutyryl cyclic AMP-induced differentiation of F9 cells. The cellular growth rate of F9 cells as determined by [3H]thymidine uptake and parallel cell counting decreased markedly during their induction with retinoic acid and dibutyryl cyclic AMP. Taken together, it is apparent that the expression of the CAD gene is cell-growth-dependent and its regulation in this system is at the posttranscriptional level.
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PMID:Posttranscriptional regulation of the expression of CAD gene during differentiation of F9 teratocarcinoma cells by induction with retinoic acid and dibutyryl cyclic AMP. 289 7

The interaction between Escherichia coli carbamoyl-phosphate synthetase (CPS) and a fluorescent analogue of an allosteric effector molecule, 1,N6-ethenoadenosine 5'-monophosphate (epsilon-AMP), has been detected by using fluorescence techniques and kinetic measurements. From fluorescence anisotropy titrations, it was found that epsilon-AMP binds to a single site on CPS with Kd = 0.033 mM. The nucleotide had a small activating effect on the rate of synthesis of carbamoyl phosphate but had no effect on the Km for ATP. To test whether epsilon-AMP binds to an allosteric site, allosteric effectors (UMP, IMP, and CMP), known to bind at the UMP/IMP site, were added to solutions containing the epsilon-AMP-CPS complex. With addition of these effector molecules, a progressive decrease of the fluorescence anisotropy was observed, indicating that bound epsilon-AMP was displaced by the allosteric effectors examined. From these titrations, the dissociation constants for UMP, IMP, CMP, ribose 5-phosphate, 2-deoxyribose 5-phosphate, and orthophosphate were determined. When MgATP, a substrate, was employed as a titrant, the observed decrease in anisotropy was consistent with the formation of a ternary complex (epsilon-AMP-CPS-MgATP). The effect of ATP binding, monitored at the allosteric site, was magnesium dependent, and free magnesium in solution was required to obtain a hyperbolic binding isotherm. Solvent accessibility of epsilon-AMP in binary (epsilon-AMP-CPS) and ternary (epsilon-AMP-CPS-MgATP) complexes was determined from acrylamide quenching, showing that the base of epsilon-AMP is well shielded from the solvent even in the presence of MgATP.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Interactive binding between the substrate and allosteric sites of carbamoyl-phosphate synthetase. 306 27

Foetal hepatocytes obtained from rats at different stages were cultured in order to investigate the inducibility of the five urea-cycle enzymes by glucagon and dibutyryl cyclic AMP (Bt2cAMP). When 18.5-day-old hepatocytes were cultured for 3 days with 10(-7) M glucagon, the activities of carbamoyl phosphate synthetase (CPS), argininosuccinase (ASL) and arginase were increased by 1.4-, 1.8- and 1.9-fold, respectively, as compared to controls. These effects were mimicked by 10(-4) M Bt2cAMP, but the activities of ornithine transcarbamylase (OTC) and argininosuccinate synthetase (ASS) were never changed by the addition of these compounds. Hepatocytes cultured at earlier stages were not responsive to glucagon unless dexamethasone was added simultaneously, suggesting that this steroid might induce some steps necessary for glucagon action. Bt2cAMP was effective as early as day 16.5 without requiring the presence of steroids. In addition, the effect of the cyclic nucleotide appeared additive or synergistic with that of dexamethasone. The simultaneous addition of actinomycin D did not affect the glucagon-induced increase in enzyme levels, thus suggesting a post-transcriptional effect of the hormone on the foetal enzyme activities. Insulin itself did not have any effect on the basal level of the enzyme activities and had only a moderate inhibitory effect on glucagon-induced ASL activity. This slight effect of insulin is in contrast with the marked inhibitory effect of dexamethasone on this enzyme activity that we described previously.
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PMID:Induction of the five urea-cycle enzymes by glucagon in cultured foetal rat hepatocytes. 332 26

We have examined the domain organization, and the locations of the sites phosphorylated by the cyclic-AMP-dependent protein kinase, in the multifunctional polypeptide of the pyrimidine-biosynthetic protein, CAD. Fragments produced after limited proteolysis by elastase or trypsin were separated by SDS/polyacrylamide gel electrophoresis and transferred onto nitrocellulose. The blots were probed with antibodies raised against the core aspartate carbamoyltransferase (ACTase) and dihydroorotase (DHOase) fragments to locate fragments containing these domains, and we also examined the locations of the phosphorylation sites by complete tryptic digestion of blotted, 32P-labelled fragments, followed by analytical isoelectric focussing. Our results are consistent with the domain order glutaminase(GLNase)-carbamoyl-phosphate synthetase-(CPSase)-DHOase-ACTase, as suggested by recently reported homologies between the predicted amino acid sequence for the Drosophila rudimentary gene product, and monofunctional CPSases/ACTases/DHOases. In particular, the finding of a 95-kDa elastase fragment which cross-reacted with both anti-DHOase and anti-ACTase antibodies rules out the previously suggested domain order: DHOase-GLNase-CPSase-ACTase. Phosphorylation by cyclic-AMP-dependent protein kinase accelerates cleavage of native CAD by both elastase and trypsin, and abolishes the protective effect of UTP. Site 1 is located close to the C-terminal end of the 160-kDa GLNase/CPSase region. Comparison with the predicted amino acid sequence of the Drosophila rudimentary gene revealed a strong homology between the tryptic peptide containing site 1 from hamster CAD, and a region at the extreme C-terminal end of the CPSase II domain of the Drosophila enzyme. Alignment of the Drosophila sequence and that of rat liver CPSase I, which is not phosphorylated by cyclic-AMP-dependent protein kinase, revealed that this putative site 1 region is missing in CPSase I. Site 2 could not be located with certainty, either from the limited proteolysis data, or from comparison of the sequence around this site and the sequence of the rudimentary gene. There were also one or more previously undetected minor phosphorylation site(s) located in the protease-sensitive hinge region between the DHOase and ACTase domains.
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PMID:Mapping of catalytic domains and phosphorylation sites in the multifunctional pyrimidine-biosynthetic protein CAD. 334 46

Cell growth using homocysteine as a source of cysteine-sulphur requires two enzymes, cystathionine synthase (CS) and gamma-cystathionase (CT). The second of these enzymes, CT, is apparently present in most cell lines regardless of their tissues of origin, since most cells can grow in vitro in the absence of cystine if they are provided with cystathionine, the intermediate in the pathway. Likewise, homocysteine will support the growth of many human cells. However, of a wide range of rodent cells, only well-differentiated rat hepatoma cells were found to grow using homocysteine in place of cystine. It is shown that cell growth in homocysteine-medium correlates well with the presence in the cells of detectable levels of CS. Furthermore, in cells able to grow in homocysteine-medium, it is possible to demonstrate the homocysteine-dependent trans-sulphuration of serine to cysteine. Growth in homocysteine-medium is not dependent on the release of preformed cysteine from disulphide complexes with serum proteins. In cell hybrids, and in 'dedifferentiated' variants of rat hepatomas, CS, but not CT, is subject to extinction coordinately with well-characterized liver-specific traits. For rodent cells, homocysteine-medium thus acts as a selective medium requiring the expression of a single liver-specific trait, CS. In addition it is shown that, in certain hepatoma variants, CS is regulated co-ordinately with a urea-cycle enzyme (carbamoyl phosphate synthetase I) by glucocorticoids and cyclic-AMP. Cell death through cysteine starvation is briefly considered. The immediate cause of death is apparently an insufficient supply of reduced glutathione. Selenium and vitamin E assist cell growth when the supply of cysteine is limiting.
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PMID:Characterization of cystathionine synthase as a selectable, liver-specific trait in rat hepatomas. 379 84

Carbamoyl-phosphate synthase gene expression is found to be primarily regulated by conditions that enhance hepatic glucocorticosteroid levels (hormone injections) and cyclic AMP levels (induction of diabetes). After birth, changes in the level of carbamoyl-phosphate synthase protein follow changes in the level of carbamoylphosphate synthase mRNA, suggesting a pretranslational control mechanism. In fetal rats, carbamoyl-phosphate synthase gene expression is regulated by the same factors as in adults. However, both the level to which carbamoyl-phosphate synthase mRNA can accumulate and the extent to which mRNA can be translated appear to be limited, indicating control mechanisms at the pretranslational and translational level. Finally, in the immediate postnatal period, a transient but pronounced decrease in the rate of degradation of carbamoyl-phosphate synthase protein may play a role in the accumulation of the enzyme.
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PMID:Developmental and hormonal regulation of carbamoyl-phosphate synthase gene expression in rat liver: evidence for control mechanisms at different levels in the perinatal period. 394 35

A study of the product-inhibition patterns of carbamoyl phosphate synthetase from bovine liver is reported. Inhibition by adenosine, AMP and inorganic ions is also reported. The results are in agreement with the previously proposed model in which the order of substrate binding is ATPMg, followed by HCO(3) (-), ATPMg and NH(4) (+). The order of product release on the basis of the reported results is carbamoyl phosphate, followed by ADPMg, ADPMg and inorganic phosphate.
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PMID:Product inhibition studies on bovine liver carbamoyl phosphate synthetase. 437 8

1. Carbamoyl phosphate synthetase activity of Phaseolus aureus extracts was assayed by coupling it to the catalytic subunit of Escherichia coli aspartate transcarbamoylase and determining the [(14)C]carbamoylaspartate so formed. The stability of the activity was improved by the addition of ornithine and dimethyl sulphoxide to the extraction medium. 2. The synthetase activity was found to utilize either glutamine or ammonia as amino donor, the Michaelis constants being 0.17+/-0.03mm and 6.1+/-1.0mm respectively. N-Acetylglutamate did not significantly alter the rate with either substrate, and azaserine inhibited the reaction with both amino donors to the same extent. 3. Ornithine was shown to stimulate the activity, and to counteract inhibition by UMP. The purine nucleotides IMP and GMP enhanced carbamoyl phosphate formation, whereas AMP had an inhibitory effect. 4. The Michaelis constant for carbamoyl phosphate was determined in concentrated extracts for both aspartate transcarbamoylase and ornithine transcarbamoylase activities, and was 0.13+/-0.03mm and 1.58+/-0.16mm respectively. The ratio of the activities of these two enzymes, determined at near-saturating substrate concentrations, was 1:3 (aspartate transcarbamoylase/ornithine transcarbamoylase). 5. It is concluded that in this plant tissue there is one enzyme, carbamoyl phosphate synthetase, supplying carbamoyl phosphate to both the pyrimidine and arginine pathways, that the pyrimidine pathway claims most of the available carbamoyl phosphate (depending on the concentration of the nucleotide effectors) when this intermediate is present at low concentrations; and that when the carbamoyl phosphate concentration is increased, possibly by ornithine stimulation, a larger proportion can be taken up by the arginine pathway.
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PMID:Pyrimidine nucleotide biosynthesis in Phaseolus aureus. Enzymic aspects of the control of carbamoyl phosphate synthesis and utilization. 457 94


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