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)

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

The purpose of this study was to clarify how changes in acid-base balance influence the rate of urea synthesis in vivo. Since ureagenesis was increased by an ammonium infusion into rats, regulation seemed to be a function of the blood ammonium concentration. The rate of urea synthesis was constant at a fixed rate of ammonium infusion and independent of the conjugate base infused, chloride or bicarbonate. The steady-state blood ammonium concentration was higher in the rats that developed metabolic acidosis. Thus it appeared that regulation was not directly mediated by this ammonium concentration per se. The rate of urea synthesis was also independent of the blood pH. Accordingly, the rate of urea synthesis was examined as a function of the plasma NH3 concentration. The rate of ureagenesis was found to be directly proportional to the plasma NH3 concentration. Assuming that plasma NH3 levels reflect those in mitochondria, the NH3 concentration yielding half-maximal rates of urea synthesis (close to 2 microM) was in the same range as Km for the rate-limiting step in ureagenesis, carbamoyl phosphate synthetase (EC 6.3.4.16). These results suggest that, at a constant ammonium concentration, the decreased rate of ureagenesis caused by a pH fall in vitro could reflect an acidosis-induced decline in the concentration of true substrate (NH3) for this pathway.
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PMID:Regulation of urea synthesis by acid-base balance in vivo: role of NH3 concentration. 381 36

The inhibition of cytosolic carbamoyl-phosphate synthetase II by acivicin was used to study the role of the cytosolic carbamoyl phosphate pool as the exclusive substrate source for de novo pyrimidine synthesis in rat hepatocytes. De novo pyrimidine synthesis was stimulated: 1. by uridine triphosphate deficiency (incubation with D-galactosamine) leading to a stimulation of cytosolic carbamoyl phosphate synthesis, and 2. by accumulation and efflux of mitochondrial carbamoyl phosphate (incubation with ammonium ions and L-norvaline). The stimulated orotate formation from cytosolic carbamoyl phosphate in UTP depleted cells was completely blocked by acivicin. It was not influenced by an inhibition of mitochondrial carbamoyl phosphate synthesis mediated by 4-pentenoate, since mitochondrial carbamoyl phosphate did not participate in cytosolic pyrimidine synthesis even in the presence of ammonium ion concentrations maintaining physiological rates of urea synthesis. An excess of ammonium ions led to an artificial accumulation and efflux of mitochondrial carbamoyl phosphate, which could be avoided by 4-pentenoate. The non-regulated stimulation of pyrimidine synthesis from surplus mitochondrial carbamoyl phosphate was not inhibited by acivicin. Utilization of mitochondrial carbamoyl phosphate for de novo pyrimidine synthesis presumably does not occur under physiological conditions because mitochondrial CP efflux depends on the accumulation of this metabolite in the mitochondria under experimental or pathological circumstances. Acivicin inhibition of CPS II thus cannot be bypassed by mitochondrial CP. It is suitable as inhibitor of the physiological de novo pyrimidine synthesis.
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PMID:The glutamine analog acivicin as antipyrimidine. Studies on the interrelationship between pyrimidine and urea synthesis in liver. 383 21

The activity changes of the urea-cycle enzymes were monitored in cultured foetal hepatocytes after dexamethasone and insulin treatments. Addition of dexamethasone induced the development of carbamoyl-phosphate synthetase, argininosuccinate synthetase, argininosuccinase and arginase activities as soon as day 16.5 of gestation. When insulin was added together with dexamethasone, it markedly inhibited the steroid-induced increase in carbamoyl-phosphate synthetase, argininosuccinate synthetase and argininosuccinase activities.
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PMID:Role of dexamethasone and insulin on the development of the five urea-cycle enzymes in cultured rat foetal hepatocytes. 388 87

After the urea cycle was proposed, considerable efforts were put forth to identify critical intermediates. This was then followed by studies of dietary and nutritional control of urea cycle enzyme activity and allosteric effectors of urea cycle enzymes. Correlation of urea cycle enzyme activity with isolated cell experiments indicated conditions where enzyme activity would be rate limiting. At physiological levels of ammonia the activation of carbamoyl-phosphate synthetase (EC 6.3.4.16) by N-acetylglutamate (NAG) is important. Various levels of NAG corresponded well with changes in the rate of citrulline and urea synthesis. Arginine was found to be an allosteric activator of N-acetylglutamate synthetase (EC 2.3.1.1). Therefore, it was possible that the rate of carbamoyl phosphate synthesis was dependent on the level of urea cycle intermediates, particularly arginine. Evidence for arginine in the regulation of NAG synthesis is not as clear as for NAG on carbamoyl phosphate synthetase I. The concentration of hepatic arginine is not necessarily an indication of the mitochondrial concentration. Only mitochondrial arginine stimulates the N-acetylglutamate synthetase. Recent studies indicate that the mitochondrial concentration of arginine is higher than the cytosolic concentration and is well above the Ka for N-acetylglutamate synthetase. Therefore, it appears that changes in arginine concentration are not physiologically important in regulating levels of NAG. However, it is possible that responses to the effector may vary with time after eating, and it may be this responsiveness that controls the level of NAG and thereby urea synthesis.
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PMID:Nutritional influences on the distribution of the urea cycle: intermediates in isolated hepatocytes. 388 33

Reuber hepatoma H-35 cells actively synthesize the urea cycle enzyme, carbamoyl-phosphate synthetase I. Treatment of H-35 cells with dexamethasone (0.14 microM), however, enhanced synthesis of the enzyme (as measured by incorporation of [35S]methionine) by 4-5-fold. Insulin (0.18 microM) completely inhibited dexamethasone-dependent stimulation of enzyme synthesis. In vitro translation and cDNA hybridization assays were employed to measure effects of dexamethasone plus or minus insulin on levels of mRNA encoding the biosynthetic precursor of carbamoyl-phosphate synthetase I (pCPS) in Reuber H-35 cells. Both measurements yielded similar results: dexamethasone increased pCPS mRNA levels by 4-5-fold and insulin suppressed this response, but only by 50%. Specific cDNA hybridization assays also demonstrated that Reuber H-35 cells, even after hormone treatments, contain only very low levels of albumin mRNA, and no detectable ornithine carbamoyl-transferase mRNA.
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PMID:Expression of carbamoyl-phosphate synthetase I mRNA in Reuber hepatoma H-35 cells. Regulation by glucocorticoid and insulin. 389 Sep 50

In isolated perfused rat liver, urea synthesis from ammonium ions was dependent on extracellular HCO3- and CO2 concentrations when the HCO3-/CO2 ratio in the influent perfusate was constant (pH 7.4). Urea synthesis was half-maximal at HCO3- = 4 mM, CO2 = 0.19 mM and was maximal at HCO3- and CO2 concentrations above 20 mM and 0.96 mM, respectively. At physiological HCO3- (25 mM) and CO2 (1.2 mM) concentrations in the influent perfusate, acetazolamide, the inhibitor of carbonic anhydrase, inhibited urea synthesis from ammonium ions (1 mM) by 50-60% and led to a 70% decrease in citrulline tissue levels. Acetazolamide concentrations required for maximal inhibition of urea synthesis were 0.01-0.1 mM. At subphysiological HCO3- and CO2 concentrations, inhibition of urea synthesis by acetazolamide was increased up to 90%. Inhibition of urea synthesis by acetazolamide was fully overcome in the presence of unphysiologically high HCO3- and CO2 concentrations, indicating that the inhibitory effect of acetazolamide is due to an inhibition of carbonic-anhydrase-catalyzed HCO3- supply for carbamoyl-phosphate synthetase, which can be bypassed when the uncatalyzed intramitochondrial HCO3- formation from portal CO2 is stimulated in the presence of high portal CO2 concentrations. With respect to HCO3- supply of mitochondrial carbamoyl-phosphate synthetase, urea synthesis can be separated into a carbonic-anhydrase-dependent (sensitive to acetazolamide at 0.5 mM) and a carbonic-anhydrase-independent (insensitive to acetazolamide) portion. Carbonic-anhydrase-independent urea synthesis linearly increased with the portal 'total CO2 addition' (which was experimentally determined to be CO2 addition plus 0.036 HCO3- addition) and was independent of the perfusate pH. At a constant 'total CO2 addition', carbonic-anhydrase-dependent urea synthesis was strongly affected by perfusate pH and increased about threefold when the perfusate pH was raised from 6.9 to 7.8. It is concluded that the pH dependent regulation of urea synthesis is predominantly due to mitochondrial carbonic anhydrase-catalyzed HCO3- supply for carbamoyl phosphate synthesis, whereas there is no control of urea synthesis by pH at the level of the five enzymes of the urea cycle. Because HCO3- provision for carbamoyl phosphate synthetase increases with increasing portal CO2 concentrations even in the absence of carbonic anhydrase activity, susceptibility of ureogenesis to pH decreases with increasing portal CO2 concentrations. This may explain the different response of urea synthesis to chronic metabolic and chronic respiratory acidosis in vivo.
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PMID:Hepatic urea synthesis and pH regulation. Role of CO2, HCO3-, pH and the activity of carbonic anhydrase. 393 68

Control of urea synthesis was studied in rat hepatocytes incubated with physiological mixtures of amino acids in which arginine was replaced by equimolar amounts of ornithine. The following observations were made. Intramitochondrial carbamoyl phosphate was always below 0.1 mM. Only when ornithine was absent and when, in addition, the concentration of amino acids was higher than four times their plasma concentration, intramitochondrial carbamoyl phosphate rose up to about 3 mM; under these conditions ammonia accumulated in the medium. The relationship between ornithine-cycle flux and the concentration of the cycle intermediates at varying amino acid concentration indicated that under near-physiological conditions the ornithine-cycle enzymes are far from being saturated with their subsidiaries. Moderate concentrations of norvaline had no effect on the rate of urea synthesis unless the cells were severely depleted of ornithine. Activation of carbamoyl-phosphate synthetase (ammonia) by addition of N-carbamoylglutamate only slightly stimulated urea production at all amino acid concentrations. However, in the presence of the activator the curve relating ornithine-cycle flux to the steady-state ammonia concentration was shifted to lower concentrations of ammonia. The intramitochondrial concentration of carbamoyl phosphate in rat liver in vivo was below 0.1 mM. This value is far below the concentration required for substantial inhibition of carbamoyl-phosphate synthetase. It is concluded that in vivo the function of activity changes in carbamoyl-phosphate synthetase, via the well-documented alterations in the intramitochondrial concentration of N-acetylglutamate, is to buffer the intrahepatic ammonia concentration rather than to affect urea production per se. At constant concentration of ammonia the rate of urea production is entirely controlled by the activity of carbamoyl-phosphate synthetase.
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PMID:Control of ureogenesis. 397 93

Urea excretion per gram of liver was increased 219% 2-5 h post-partial hepatectomy (Hx) and 45% 24-27 h post-Hx. Mitochondrial carbamoyl-phosphate synthetase was also increased 5 h post-Hx but was not increased at 27 h. An NH4+ load did not increase urea excretion per gram liver or the enzyme activity noticeably in the 2- to 5-h period but did increase them 24-27 h post-Hx. These results suggest that the enzyme activity and urea formation per unit weight of liver were nearly maximal early during regeneration. Orotic acid excretion per gram of liver in rats that received NH4+ was increased more than 30-fold 2-5 h post-Hx and was similar in this respect to nonhepatectomized rats. Ornithine prevented the increase in both normal and hepatectomized rats, suggesting that ornithine was rate limiting for the ornithine carbamoyltransferase (OCT) reaction. The orotic acid excretion response to NH4+ was much less 24-27 h post-Hx, indicating that ornithine availability for the OCT reaction may be increased at this time.
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PMID:Carbamoyl-phosphate synthetase I activity and ureagenesis in regenerating liver of the normal rat. 399 79

Experiments with carbamoyl phosphate synthetase (ammonia) in solution and in isolated mitochondria are reported which show the following. NH3 rather than NH4+ is the substrate of the enzyme. The apparent Km of NH3 for the purified enzyme is about 38 microM. The apparent Km for NH3 measured in intact isolated mitochondria is about 13 microM. This value was obtained for both coupled and uncoupled mitochondria and was unchanged when the rate of carbamoyl phosphate synthesis was increased 2-fold by incubating uncoupled mitochondria in the presence of 5 mM-N-acetylglutamate. According to the literature, the concentration of NH3 in liver is well below the measured apparent Km. On the basis of this and previous work we conclude that, quantitatively, changes in liver [NH3] and [ornithine] are likely to be the most important factors in the fast regulation of synthesis of carbamoyl phosphate and urea. This conclusion is consistent with all available evidence obtained with isolated mitochondria, isolated hepatocytes, perfused liver and whole animals.
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PMID:The apparent Km of ammonia for carbamoyl phosphate synthetase (ammonia) in situ. 403 55


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