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)

The role of residues of the ornithine activator site is probed by mutagenesis in Escherichia coli carbamoyl phosphate synthetase (CPS). Mutations E783A, E783L, E892A and E892L abolish ornithine binding, E783D and T1042V decrease 2-3 orders of magnitude and E892D decreased 10-fold apparent affinity for ornithine. None of the mutations inactivates CPS. E783 mutations hamper carbamate phosphorylation and increase K(+) and MgATP requirements, possibly by perturbing the K(+)-loop near the carbamate phosphorylation site. Mutation E892A activates the enzyme similarly to ornithine, possibly by altering the position of K891 at the opening of the tunnel that delivers the carbamate to its phosphorylation site. T1042V also influences modulation by IMP and UMP, supporting signal transmission from the nucleotide effector to the ornithine site mediated by a hydrogen bond network involving T1042. Ornithine activation of CPS may be mediated by K(+)-loop and tunnel gating changes.
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PMID:Mechanism of allosteric modulation of Escherichia coli carbamoyl phosphate synthetase probed by site-directed mutagenesis of ornithine site residues. 1194 74

Although carbamoyl-phosphate synthetases (CPSs) share sequence identity, multidomain structure, and reaction mechanism, they have varying physiological roles and allosteric effectors. Escherichia coli CPS (eCPS) provides CP for both arginine and pyrimidine nucleotide biosynthesis and is allosterically regulated by metabolites from both pathways, with inhibition by UMP and activation by IMP and ornithine. The arginine-specific CPS from Saccharomyces cerevisiae (sCPS), however, apparently responds to no allosteric effectors. We have designed and analyzed a chimeric CPS (chCPS, in which the C-terminal 136 residues of eCPS were replaced by the corresponding residues of sCPS) to define the structural basis for the allosteric nonresponsiveness of sCPS and thereby provide insight into the mechanism for allosteric selectivity and responsiveness in the other CPSs. Surprisingly, ornithine and UMP each had a significant effect on chCPS activity, and did so at concentrations that were similar to those effective for eCPS. We further found that sCPS bound both UMP and IMP and that chCPS bound IMP, although none of these interactions led to changes in enzymatic activity. These findings strongly suggest that the nonresponsive sCPS is not able to communicate occupancy of the allosteric site to the active site but does contain a latent allosteric interaction domain.
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PMID:Unmasking a functional allosteric domain in an allosterically nonresponsive carbamoyl-phosphate synthetase. 1224 18

The fully grown but nonmetamorphosed (juvenile) axolotl Ambystoma mexicanum was ureogenic and primarily ureotelic in water. A complete ornithine-urea cycle (OUC) was present in the liver. Aerial exposure impeded urea (but not ammonia) excretion, leading to a decrease in the percentage of nitrogen excreted as urea in the first 24 h. However, urea and not ammonia accumulated in the muscle, liver, and plasma during aerial exposure. By 48 h, the rate of urea excretion recovered fully, probably due to the greater urea concentration gradient in the kidney. It is generally accepted that an increase in carbamoyl phosphate synthetase activity is especially critical in the developmental transition from ammonotelism to ureotelism in the amphibian. Results from this study indicate that such a transition in A. mexicanum would have occurred before migration to land. Aerial exposure for 72 h exhibited no significant effect on carbamoyl phosphate synthetase-I activity or that of other OUC enzymes (with the exception of ornithine transcarbamoylase) from the liver of the juvenile A. mexicanum. This supports our hypothesis that the capacities of OUC enzymes present in the liver of the aquatic juvenile axolotl were adequate to prepare it for its invasion of the terrestrial environment. The high OUC capacity was further supported by the capability of the juvenile A. mexicanum to survive in 10 mM NH(4)Cl without accumulating amino acids in its body. The majority of the accumulating endogenous and exogenous ammonia was detoxified to urea, which led to a greater than twofold increase in urea levels in the muscle, liver, and plasma and a significant increase in urea excretion by hour 96. Hence, it can be concluded that the juvenile axolotl acquired ureotelism while submerged in water, and its hepatic capacity of urea synthesis was more than adequate to handle the toxicity of endogenous ammonia during migration to land.
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PMID:Excretory nitrogen metabolism in the juvenile axolotl Ambystoma mexicanum: differences in aquatic and terrestrial environments. 1252 47

The white-edge freshwater whip ray Himantura signifer can survive in freshwater (0.7 per thousand ) indefinitely or in brackish water (20 per thousand ) for at least two weeks in the laboratory. In freshwater, the blood plasma was maintained hyperosmotic to that of the external medium. There was approximately 44 mmol l(-1) of urea in the plasma, with the rest of the osmolality made up mainly by Na(+) and Cl(-). In freshwater, it was not completely ureotelic, excreting up to 45% of its nitrogenous waste as urea. Unlike the South American freshwater stingray Potamotrygon motoro, H. signifer has a functional ornithine-urea cycle (OUC) in the liver, with hepatic carbamoylphosphate synthetase III (CPS III) and glutamine synthetase (GS) activities lower than those of the marine blue-spotted fan tail ray Taeniura lymma. More importantly, the stomach of H. signifer also possesses a functional OUC, the capacity (based on CPS III activity) of which was approximately 70% that in the liver. When H. signifer was exposed to a progressive increase in salinity through an 8-day period, there was a continuous decrease in the rate of ammonia excretion. In 20 per thousand water, urea levels in the muscle, brain and plasma increased significantly. In the plasma, osmolality increased to 571 mosmol kg(-1), in which urea contributed 83 mmol l(-1). Approximately 59% of the excess urea accumulated in the tissues of the specimens exposed to 20 per thousand water was equivalent to the deficit in ammonia excretion through the 8-day period, indicating that an increase in the rate of urea synthesis de novo at higher salinities would have occurred. Indeed, there was an induction in the activity of CPS III in both the liver and stomach, and activities of GS, ornithine transcarbamoylase and arginase in the liver. Furthermore, there was a significant decrease in the rate of urea excretion during passage through 5 per thousand, 10 per thousand and 15 per thousand water. Although the local T. lymma in full-strength sea water (30 per thousand ) had a much greater plasma urea concentration (380 mmol l(-1)), its urea excretion rate (4.7 micromol day(-1) g(-1)) was comparable with that of H. signifier in 20 per thousand water. Therefore, H. signifer appears to have reduced its capacity to retain urea in order to survive in the freshwater environment and, consequently, it could not survive well in full-strength seawater.
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PMID:The osmotic response of the Asian freshwater stingray (Himantura signifer) to increased salinity: a comparison with marine (Taeniura lymma) and Amazonian freshwater (Potamotrygon motoro) stingrays. 1287 62

Like the marine ray Taeniura lymma, the African lungfish Protopterus dolloi possesses carbamoyl phosphate III (CPS III) in the liver and not carbamoyl phosphate I (CPS I), as in the mouse Mus musculus or as in other African lungfish reported elsewhere. However, similar to other African lungfish and tetrapods, hepatic arginase of P. dolloi is present mainly in the cytosol. Glutamine synthetase activity is present in both the mitochondrial and cytosolic fractions of the liver of P. dolloi. Therefore, we conclude that P. dolloi is a more primitive extant lungfish, which is intermediate between aquatic fish and terrestrial tetrapods, and represents a link in the fish-tetrapod continuum. During 6 days of aerial exposure, the ammonia excretion rate in P. dolloi decreased significantly to 8-16% of the submerged control. However, there were no significant increases in ammonia contents in the muscle, liver or plasma of specimens exposed to air for 6 days. These results suggest that (1). endogenous ammonia production was drastically reduced and (2). endogenous ammonia was detoxified effectively into urea. Indeed, there were significant decreases in glutamate, glutamine and lysine levels in the livers of fish exposed to air, which led to a decrease in the total free amino acid content. This indirectly confirms that the specimen had reduced its rates of proteolysis and/or amino acid catabolism to suppress endogenous ammonia production. Simultaneously, there were significant increases in urea levels in the muscle (8-fold), liver (10.5-fold) and plasma (12.6-fold) of specimens exposed to air for 6 days. Furthermore, there was an increase in the hepatic ornithine-urea cycle (OUC) capacity, with significant increases in the activities of CPS III (3.8-fold), argininosuccinate synthetase + lyase (1.8-fold) and, more importantly, glutamine synthetase (2.2-fold). This is the first report on the upregulation of OUC capacity and urea synthesis rate in an African lungfish exposed to air. Upon re-immersion, the urea excretion rate increased 22-fold compared with that of the control specimen, which is the greatest increase among fish during emersion-immersion transitions and suggests that P. dolloi possesses transporters that facilitate the excretion of urea in water.
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PMID:Urea synthesis in the African lungfish Protopterus dolloi--hepatic carbamoyl phosphate synthetase III and glutamine synthetase are upregulated by 6 days of aerial exposure. 1296 53

The white-edge whip tail ray Himantura signifer inhabits a freshwater environment but has retained the capability to synthesize urea de novo through the arginine-ornithine-urea cycle (OUC). The present study aimed to elucidate whether the capacity of urea synthesis in H. signifer could be upregulated in response to environmental ammonia exposure. When H. signifer was exposed to environmental ammonia, fairly high concentrations of ammonia were accumulated in the plasma and other tissues. This would subsequently reduce the net influx of exogenous ammonia by reducing the NH(3) partial pressure gradient across the branchial and body surfaces. There was also an increase in the OUC capacity in the liver. Since the ammonia produced endogenously could not be excreted effectively in the presence of environmental ammonia, it was detoxified into urea through the OUC. In comparison, the South American freshwater stingray Potamotrygon motoro, which has lost the capability to synthesize urea de novo, was unable to detoxify ammonia to urea during ammonia loading. No increase in glutamine was observed in the various tissues of H. signifer exposed to environmental ammonia despite a significant increase in the hepatic glutamine synthetase activity. These results indicate that the excess glutamine formed was channelled completely into urea formation through carbamoyl phosphate synthetase III. It has been reported elsewhere that both urea synthesis and urea retention were upregulated in H. signifer exposed to 20 per thousand water for osmoregulatory purposes. By contrast, for H. signifer exposed to environmental ammonia in freshwater, the excess urea formed was excreted to the external medium instead. This suggests that the effectiveness of urea synthesis de novo as a strategy to detoxify ammonia is determined not simply by an increase in the capacity of urea synthesis but, more importantly, by the ability of the animal to control the direction (i.e. absorption or excretion) and rate of urea transport. Our results suggest that such a strategy began to develop in those elasmobranchs, e.g. H. signifer, that migrate into a freshwater environment from the sea but not in those permanently adapted to a freshwater environment.
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PMID:A comparison of the effects of environmental ammonia exposure on the Asian freshwater stingray Himantura signifer and the Amazonian freshwater stingray Potamotrygon motoro. 1296 54

1. Carbamoyl phosphate synthetase, ornithine transcarbamoylase, the arginine-synthetase system and arginase were measured in the livers of ammoniotelic, ureotelic and uricotelic animals. The chelonian reptiles, whose nitrogen excretory patterns vary according to the habitat, and the Mexican axolotl, a neotenic species, were also studied. 2. The levels of the activities of the first three enzymes mentioned correlate with the amount of nitrogen excreted as urea. 3. The terrestrial turtle, which excretes mainly uric acid, maintains a high arginase activity but has very low levels of the activities of the other three enzymes. 4. The first three enzymes of the urea cycle vary in the phylogenic scale in a co-ordinated manner, which suggests that they are under the same regulatory mechanism. 5. Urea formation from endogenous arginine in vitro has a low efficiency in the Mexican axolotl. 6. The induction of metamorphosis in the Mexican axolotl by the administration of l-tri-iodothyronine, which causes a shift from ammonio-ureotelism to complete ureotelism, is accompanied by an increase mainly in carbamoyl phosphate synthetase and also by an improvement in the efficiency of hydrolysis of endogenous arginine in vitro to give urea. 7. The results obtained by differential centrifugation of the urea-cycle enzymes in rat and Mexican-axolotl livers are presented. The location requirements for the integration of a metabolic cycle are discussed.
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PMID:THE REGULATION OF UREA-BIOSYNTHESIS ENZYMES IN VERTEBRATES. 1434 46

1. The activities of enzymes of the urea cycle [carbamoyl phosphate synthetase, ornithine transcarbamoylase, argininosuccinate synthetase, argininosuccinase (these last two comprising the arginine-synthetase system) and arginase] have been measured in control, alloxan-diabetic and glucagon-treated rats. In addition, measurements were made on alloxan-diabetic rats treated with protamine-zinc-insulin. 2. Treatment of rats with glucagon for 3 days results in a marked increase in the activities of three enzymes of the urea cycle (carbamoyl phosphate synthetase, argininosuccinate synthetase and argininosuccinase). The pattern of change in the alloxan-diabetic group is very similar to that of the glucagon-treated group, although the magnitude of the change was much greater. 3. Comparison was made of the actual and potential rate of urea synthesis in normal and diabetic rats. In both groups the potential rate of urea production, as measured by the activity of the rate-limiting enzyme, argininosuccinate synthetase, slightly exceeds the actual rate of synthesis by liver slices in the presence of substrates. The relative activities of the actual and potential rates were similar in the two groups of animals, this ratio being 1:0.70. 4. In the alloxan-diabetic rats treated with protamine-zinc-insulin for 2.5 or 4 days there was a marked increase in liver weight. This was associated with a rise in the total hepatic activity of the urea-cycle enzymes located in the soluble fraction of the cell (the arginine-synthetase system and arginase) after 2.5 days of treatment. After 4 days of treatment the concentration of these enzymes/g. of liver decreased, and the total hepatic content then reverted to the untreated alloxan-diabetic value. 5. No effects of glucagon or of insulin in vitro could be found on the rate of urea production by liver slices. 6. The present results are discussed in relation to how far this pattern of change is typical of conditions resulting in a high urea output, and comparison has been made with other values in the literature.
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PMID:INFLUENCE OF PANCREATIC HORMONES ON ENZYMES CONCERNED WITH UREA SYNTHESIS IN RAT LIVER. 1434 1

The Chinese fire-belly newt Cynops orientalis reverts to an aquatic mode of living when sexually mature. Despite living in water, sexually mature C. orientalis maintained high capacity for hepatic urea synthesis. However, it had a lower rate of urea production than other terrestrial amphibians because endogenous ammonia could diffuse out to the external medium as NH3. This conserves cellular energy because urea synthesis is energetically expensive. Simultaneously, C. orientalis also reduced the rate of urea excretion, and excreted 33% of the total nitrogenous waste as ammonia. Upon exposure to land, C. orientalis increased the rate of urea synthesis from accumulating endogenous ammonia. The increased rate of urea synthesis was within the inherent capacity of the hepatic ornithine-urea cycle; there was no induction of hepatic carbamoyl phosphate synthetase or ornithine transcarbamoylase activities and there was no reduction in ammonia production. When exposed to water containing 75 mmol.l(-1) NH4Cl, the rates of both urea synthesis and urea excretion increased. Under such experimental conditions, the ornithine-urea cycle may be operating close to its limit; glutamine began to accumulate in the body, and endogenous ammonia production via amino acid catabolism was reduced.
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PMID:Excretory nitrogen metabolism in the Chinese fire-belly newt Cynops orientalis in water, on land, or in high concentrations of environmental ammonia. 1461 Jun 82

CPSase (carbamoyl-phosphate synthetase II), a component of CAD protein (multienzymic protein with CPSase, aspartate transcarbamylase and dihydro-orotase activities), catalyses the regulated steps in the de novo synthesis of pyrimidines. Unlike the orthologous Escherichia coli enzyme that is regulated by UMP, inosine monophosphate and ornithine, the mammalian CPSase is allosterically inhibited by UTP, and activated by PRPP (5-phosphoribosyl-a-pyrophosphate) and phosphorylation. Four residues (Thr974, Lys993, Lys954 and Thr977) are critical to the E. coli inosine monophosphate/UMP-binding pocket. In the present study, three of the corresponding residues in the hamster CPSase were altered to determine if they affect either PRPP activation or UTP inhibition. Substitution of the hamster residue, positionally equivalent to Thr974 in the E. coli enzyme, with alanine residue led to an enzyme with 5-fold lower activity and a near loss of PRPP activation. Whereas replacement of the tryptophan residue at position 993 had no effect, an Asp992-->Asn substitution yielded a much-activated enzyme that behaved as if PRPP was present. The substitution Lys954-->Glu had no effect on PRPP stimulation. Only modest decreases in UTP inhibitions were observed with each of the altered CPSases. The results also show that while PRPP and UTP can act simultaneously, PRPP activation is dominant. Apparently, UTP and PRPP have distinctly different associations within the mammalian enzyme. The findings of the present study may prove relevant to the neuropathology of Lesch-Nyhan syndrome
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PMID:Substitutions in hamster CAD carbamoyl-phosphate synthetase alter allosteric response to 5-phosphoribosyl-alpha-pyrophosphate (PRPP) and UTP. 1465 76


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