Physical location of the site for N-acetyl-L-glutamate, the allosteric activator of carbamoyl phosphate synthetase, in the 20-kilodalton COOH-terminal domain

Mammalian liver mitochondrial carbamoyl phosphate synthetase, a polypeptide of 160 kDa, is activated allosterically by N-acetyl-L-glutamate. The analogue of this activator N-(chloroacetyl)-L-[14C]glutamate has been found to serve as a photoaffinity label for this enzyme. The specificity was demonstrated by the drastic reduction in the radioactivity bound to the protein when (a) an excess of unlabeled acetylglutamate was present during the irradiation and (b) the enzyme was replaced by pyruvate kinase, an enzyme that is not affected by acetylglutamate. The labeling was due to the photoactivation of the chloroacetyl group since there was no labeling under equal conditions with acetyl[14C]glutamate. To localize the binding site, limited proteolysis was used. Trypsin cleaves carbamoyl phosphate synthetase into complementary NH2- and COOH-terminal fragments of about 140 and 20 kDa, respectively [Powers-Lee, S. G., & Corina, K. (1986) J. Biol. Chem. 261, 15349-15352], but only the latter was found to be labeled. Similarly, of the various fragments generated by elastase, only two, of 20 and 120 kDa, contain the COOH terminus [see Powers-Lee and Corina (1986) above] and were found to be labeled. Thus, the binding site for acetylglutamate is within 20 kDa from the COOH terminus. This excludes the possibility that the acetylglutamate binding site evolved from an ancestral substrate site for glutamine: this substrate binds to the small subunit of the Escherichia coli enzyme, which is homologous to the NH2-terminal domain of the rat liver enzyme. Exhaustive tryptic digestion of photolabeled carbamoyl phosphate synthetase yielded a single radioactive peak, suggesting that the labeling is restricted to a single minimal tryptic peptide.     

Mitochondrial carbamoyl phosphate synthetase activity in the absence of N-acetyl-L-glutamate. Mechanism of activation by this cofactor

Rat liver carbamoyl-phosphate synthetase I is shown to have synthetase and ATPase activity in the absence of acetylglutamate. Km values for ATP, Mg2+ and K+ are greatly increased, the Km for HCO-3 is not changed much, and the Km for NH+4 is markedly reduced. Vmax for the synthetase reaction is less than 20% of that of the acetylglutamate-activated enzyme whereas Vmax for the ATPase activity is greater than 40% of that with acetylglutamate. Pulse-chase experiments with H14CO-3 show formation of less "active CO2" (the central intermediate) than with acetylglutamate; ATPase activity is reduced in proportion, but the synthetase activity is much smaller. Binding of one ATP molecule with high affinity (Kd = 20-30 microM) is shown in the absence of acetylglutamate. This appears to be the molecule of ATPB (ATPB provides the phosphoryl group of carbamoyl phosphate). In contrast, the affinity for ATPA (ATPA yields Pi) is much reduced. Initial velocity measurements without acetylglutamate show a time lag before reaching a constant velocity. At 50 microM acetylglutamate the lag is much longer, but at 10 mM acetylglutamate it is shorter. Activation by acetylglutamate requires ATP at concentrations sufficient to occupy the ATPA and the ATPB binding sites. Preincubation with 10 mM acetylglutamate alone shortens the activation time. From these findings we propose an allosteric model for activation of carbamoyl-phosphate synthetase in which there are two active states, R and R . AcGlu. Binding of ATPA is associated with the conversion of T to R. R . AcGlu differs from R in that transfer to carbamate of the gamma-phosphoryl group of ATPB appears to be facilitated.     

Hepatic urea synthesis and pH regulation. Role of CO2, HCO3-, pH and the activity of carbonic anhydrase

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.     

An improved method for determination of N-acetyl-L-glutamate by its function as an activator of carbamoyl phosphate synthetase I

N-Acetyl-L-glutamate has been examined with regard to its ability to activate carbamoyl phosphate synthetase I (EC 6.3.4.16). Substance(s) inhibitory to carbamoyl phosphate synthetase, present even in the partially purified preparation of rat liver extracts, interfered with the measurement of acetylglutamate. In the experiments using chelating agents, metals were apparently involved in this inhibition. When the partially purified preparation of liver extract was placed on a Chelex 100 column, the inhibitor was eliminated and accurate measurements of acetylglutamate content could be made. Evidence supporting the validity of this improved method is given. A significant difference was observed between acetylglutamate levels determined by the present method and by the one using aminoacylase I (N-acylamino acid amidohydrolase, EC 3.5.1.14) to hydrolyze acetylglutamate followed by assay of the glutamate generated. We searched for the presence of glutamate derivatives other than acetylglutamate. When impure tissue preparations containing acetylglutamate were treated with a commercial preparation of aminoacylase, there was an excess amount of glutamate apparently derived from compounds other than acetylglutamate. This can lead to an overestimation of the tissue levels of acetylglutamate.     

Purification and properties of glutamine synthetase from liver of Squalus acanthias

Ammonia assimilation for urea synthesis by liver mitochondria in marine elasmobranchs involves, initially, formation of glutamine which is subsequently utilized for mitochondrial carbamoyl phosphate synthesis [P. M. Anderson and C. A. Casey (1984) J. Biol. Chem. 259, 456-462]. The purpose of this study was to determine if the glutamine synthetase catalyzing this first step in urea synthesis has properties uniquely related to this function. Glutamine synthetase has been highly purified from isolated liver mitochondria of Squalus acanthias, a representative elasmobranch. The purified enzyme has a molecular weight of approximately 400,000 in the presence of Mg2+, MgATP, and L-glutamate, but dissociates reversibly to a species with a molecular weight of approximately 200,000 in the absence of MgATP and L-glutamate. Association with the glutamine- and acetylglutamate-dependent carbamoyl phosphate synthetase, also located in the mitochondria, could not be demonstrated. The subunit molecular weight is approximately 46,000. The pH optimum of the biosynthesis reaction is 7.1-7.4. The purified enzyme is stabilized by MgATP and glutamate and by ethylene glycol, and is activated by 5-10% ethylene glycol. The apparent Km values for MgATP, L-glutamate, and ammonia (NH4+-NH3) are 0.7, 11.0, and 0.015 mM, respectively. Mg2+ in excess of that required to complex ATP as MgATP is required for maximal activity; Mn2+ cannot replace Mg2+. The enzyme is activated by low concentrations of chloride, bromide, or iodide; this effect appears to be related to decreases in the apparent Km for glutamate. The enzyme is inhibited by physiological concentrations of urea, but is not significantly affected by physiological concentrations of trimethylamine-N-oxide. Except for activation by halogen anions and the very low apparent Km for ammonia, this elasmobranch glutamine synthetase has properties similar to those reported for mammalian and avian glutamine synthetases. The very low apparent Km for ammonia may be specifically related to the unique role of this glutamine synthetase in mitochondrial assimilation of ammonia for urea synthesis.     

Carbonic anhydrase inhibitors. Interaction of isozymes I, II, IV, V, and IX with phosphates, carbamoyl phosphate, and the phosphonate antiviral drug foscarnet

A detailed inhibition study of five carbonic anhydrase (CA, EC 4.2.1.1) isozymes with inorganic phosphates, carbamoyl phosphate, the antiviral phosphonate foscarnet as well as formate is reported. The cytosolic isozyme hCA I was weakly inhibited by neutral phosphate, strongly inhibited by carbamoyl phosphate (K(I) of 9.4 microM), and activated by hydrogen- and dihydrogenphosphate, foscarnet and formate (best activator foscarnet, K(A)=12 microM). The cytosolic isozyme hCA II was weakly inhibited by all the investigated anions, with carbamoyl phosphate showing a K(I) of 0.31 mM. The membrane-associated isozyme hCA IV was the most sensitive to inhibition by phosphates/phosphonates, showing a K(I) of 84 nM for PO(4)(3-), of 9.8 microM for HPO(4)(2-), and of 9.9 microM for carbamoyl phosphate. Foscarnet was the best inhibitor of this isozyme (K(I) of 0.82 mM) highly abundant in the kidneys, which may explain some of the renal side effects of the drug. The mitochondrial isozyme hCA V was weakly inhibited by all phosphates/phosphonates, except carbamoyl phosphate, which showed a K(I) of 8.5 microM. Thus, CA V cannot be the isozyme involved in the carbamoyl phosphate synthetase I biosynthetic reaction, as hypothesized earlier. Furthermore, the relative resistance of CA V to inhibition by inorganic phosphates suggests an evolutionary adaptation of this mitochondrial isozyme to the presence of high concentrations of such anions in these energy-converting organelles, where high amounts of ATP are produced by ATP synthetase, from ADP and inorganic phosphates. The transmembrane, tumor-associated isozyme hCA IX was on the other hand slightly inhibited by all these anions.     

Differential effects of N-acetylglutamate on citrulline synthesis by coupled and uncoupled mitochondria

When rats were placed on a low-protein (5%) diet for 24 h or less, liver mitochondrial acetylglutamate decreased rapidly, carbamyl phosphate synthetase (ammonia) and ornithine transcarbamylase decreased little, and carbamyl phosphate synthesis (measured as citrulline) by isolated mitochondria occurred at very low rates. The matrix acetylglutamate content of these mitochondria, whether coupled or uncoupled, was increased similarly by preincubating them with added acetylglutamate, but citrulline synthesis increased from less than 1 to 2.3 nmol min-1 mg-1 in the coupled state, and from less than 1 to 35 nmol min-1 mg-1 in the uncoupled state. However, when coupled mitochondria were incubated with the substrates required for the synthesis of acetylglutamate in the matrix, citrulline synthesis increased to 48 nmol min-1 mg-1; this rate was similar to that of mitochondria from control rats (fed a normal diet). When mitochondria from controls were incubated with up to 5mM acetylglutamate, citrulline synthesis by coupled mitochondria was increased by 10 to 40%, while synthesis by uncoupled mitochondria was 1.5 to 4 times higher than that observed with the coupled mitochondria; matrix acetylglutamate in both conditions rose to levels similar to those in the medium. The reason for the different behavior of carbamyl phosphate synthetase (ammonia) in coupled and uncoupled mitochondria was not apparent; neither oxidative phosphorylation nor ornithine transport were limiting in the coupled system. These observations are an example of the restrictions imposed upon enzymatic systems by the conditions existing in the mitochondrial matrix, and of the different behavior of carbamyl phosphate synthetase in situ and in solution. In addition, they show that conclusions about the characteristics of the enzyme in coupled mitochondria based on observations made in uncoupled mitochondria are not necessarily justified.     

Carbamoyl-phosphate synthetase I. Kinetics of binding and dissociation of acetylglutamate and of activation and deactivation

The dissociation of the cofactor, acetylglutamate, from the enzyme-cofactor complex formed by carbamoyl-phosphate synthetase I of rat liver in the presence of ATP, Mg2+, K+ and HCO-3 has been studied by centrifugal gel filtration. The rate of its dissociation (k, 0.13 s-1) is considerably slower than the rate of enzyme turnover (approximately equal to 6 s-1) and it is not increased by ammonia, although ammonia reduces the rate of reassociation of the cofactor. Omission of ATP, Mg2+ or K+ from the column buffer leads to virtually complete dissociation of bound acetylglutamate during passage through the column (0.5-2 min), owing to an increase in dissociation and a decrease in reassociation, but reduction of free Mg2+ alone has the opposite action. Dilution of the enzyme-cofactor complex into a large volume of buffer causes a biphasic loss of enzyme activity with a t1/2 of the first phase comparable with that of the dissociation of acetylglutamate. These findings show (a) that acetylglutamate does not dissociate with each turnover of the enzyme; (b) that there are rapid interactions between binding of acetylglutamate and ATPA (ATPA yields Pi in the overall reaction), Mg2+ and K+, suggesting that these ligands bind in close proximity; and (c) that the enzyme transiently retains considerable activity after dissociation of the cofactor.     

Limited proteolysis reveals low-affinity binding of N-acetyl-L-glutamate to rat-liver carbamoyl-phosphate synthetase (ammonia)

Carbamoyl-phosphate synthetase was inactivated by elastase with first-order kinetics, and N-acetyl-L-glutamate speeded inactivation. From the dependence of the t1/2 value for inactivation on the concentration of acetylglutamate we estimate a Kd value for binding of the activator of 0.365 mM, which is approximately 600 times greater than in the presence of ATP, HCO3-, K+ and Mg2+. K+ and Mg2+ are not required for binding with low affinity, and in the absence of ATP they do not appear to increase the affinity for acetylglutamate. In the presence of acetylglutamate, mixtures of ATP, K+ and Mg2+ protect the enzyme from inactivation. ADP or AdoPP[NH]P partly replaced ATP in protecting the enzyme and thus binding of the nucleotide without further reaction is enough for protection. Two partial activities of the enzyme were inactivated by elastase to the same extent as the overall reaction, and thus elastase affects some property of the enzyme which is essential for catalysis. With other proteinases tested, inactivation was also accelerated by acetylglutamate and was slowed by mixtures of ATP, K+, Mg2+ and acetylglutamate, suggesting that changes in the accessibility of susceptible bonds are responsible for the changes in the degree of inactivation. It is concluded that elastase attacks at or close to the binding sites for ATP, and that exposure of the binding site for the ATP molecule that yields Pi (ATPA) upon binding of acetylglutamate causes the acceleration of the proteolytic inactivation.     

Changes in urea cycle-related metabolites in the mouse after combined administration of valproic acid and an amino acid load

Increased blood ammonia was induced in fasting mice by ip administration of 200 mg/kg Na-valproate followed 1 h later by 13 and 4 mmol/kg alanine and ornithine, respectively. When valproate was not used blood or liver ammonia was not increased, but increases were observed in liver glutamate (5-fold), glutamine (2-fold), aspartate (5-fold), acetylglutamate (15-fold), citrulline (35-fold), argininosuccinate (11-fold), arginine (11-fold), and urea (3-fold). The level of carbamoyl phosphate (less than 2 nmol/g) was, by far, the lowest of all urea cycle intermediates. The large increase in citrulline indicates that argininosuccinate synthesis was limiting, and that the increase in acetylglutamate induced a considerable activation of carbamoyl phosphate synthetase, which agrees with theoretical expectations, irrespective of the actual KD value for acetylglutamate. Pretreatment with valproate resulted in lower hepatic levels of glutamate, glutamine, aspartate, acetyl-CoA, and acetylglutamate. At the level found of acetylglutamate the activation of carbamoyl phosphate synthetase would be expected to be similar to that without valproate. Indeed, the levels of citrulline were similar with or without valproate. Argininosuccinate, arginine, and urea levels exhibited little if any change. Although the model used may not replicate exactly the situation in patients, from our results it appears that changes in citrullinogenesis or in other steps of the urea cycle do not account for the increase in blood ammonia induced by valproate, and it is proposed that valproate may alter glutamine metabolism.     

Levels of carbamoyl phosphate synthetase I in livers of young and old rats assessed by activity and immunoassays and by electron microscopic immunogold procedures

Carbamoyl phosphate synthetase I, the most abundant protein of rat liver mitochondria, plays a key role in synthesis of urea. Because aging affects some liver functions, and because there is no information on the levels of carbamoyl phosphate synthetase I during aging, we assayed the activity of this enzyme and determined immunologically the level of carbamoyl phosphate synthetase I in liver homogenates from young (4 months) and old (18 or 26 months) rats. In addition, we used electron microscopic immunogold procedures to locate and measure the amount of the enzyme in the mitochondrial matrix. There is no significant change in enzyme activity or enzyme protein content with age, although there is a higher concentration of the enzyme in the mitochondria (c. 1.5 times greater) from old rats, which is compensated by a decrease in the fractional volume of the mitochondrial compartment during aging.     

Immunoferritin location of carbamoyl phosphate synthetase in rat liver.

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.     

Inactivation of mitochondrial carbamoyl phosphate synthetase induced by ascorbate, oxygen, and Fe3+ in the presence of acetylglutamate: protection by ATP and HCO3- and lack of inactivation of ornithine transcarbamylase

Of the two mitochondrial enzymes of the urea cycle, carbamoyl phosphate synthetase (CPS) was and ornithine transcarbamylase (OTC) was not inactivated by the Fe3+-oxygen-ascorbate model system for mixed-function oxidation [R. L. Levine, (1983) J. Biol. Chem. 258, 11828-11833]. The susceptibility of OTC was not increased by its substrates, products, or inhibitors, whereas that of CPS was markedly increased by acetylglutamate (its allosteric activator) when ATP was absent. Thus, acetylglutamate binds in the absence of ATP and exposes to oxidation essential groups of the enzyme. We estimate for this binding a KD value of 1.6 mM, which greatly exceeds the KD values (less than 10 microM) determined in the presence of ATP and bicarbonate. ATP, and even more, mixtures of ATP and bicarbonate protected CPS from inactivation. Acetylglutamate exposes the site for the ATP molecule that yields Pi, and it appears that ATP protects by binding at this site. Experiments of limited proteolysis with elastase suggest that oxidation prevents this binding of ATP and show that it accelerates cleavage of CPS by the protease, thus supporting the idea that oxidation may precede proteolysis. Trypsin, chymotrypsin, and papain also hydrolyze the oxidized enzyme considerably faster than the native enzyme. Our results also support the idea that oxidative inactivation is site specific and requires sites on the enzyme for Me2+ and, possibly, for a nucleotide.     

Effects of inhibition of ornithine aminotransferase or of general aminotransferases on urea and citrulline synthesis and on the levels of acetylglutamate in isolated rat hepatocytes

Summary Canaline and gabaculine, inhibitors of γ-aminotransferases and thus of ornithine aminotransferase (E.C. 2.6.1.13), decreased the flow through ornithine carbamoyl transferase (E.C. 2.1.3.3) in isolated rat hepatocytes incubated with 10 mM NH₄Cl and ornithine. The levels of acetylglutamate, an essential activator of carbamoyl phosphate synthetase (ammonia) (E.C. 6.3.4.16), were also decreased, suggesting that the inhibitors had also caused a decrease in the rate of carbamoyl phosphate synthesis. Under these conditions, ornithine appears to be a precursor of acetylglutamate, via ornithine aminotransferase, possibly as a consequence of glutamate synthesis. The influence of aminooxyacetate, an aminotransferase inhibitor, has also been examined.     

Arginine-specific carbamoyl phosphate metabolism in mitochondria of Neurospora crassa. Channeling and control by arginine

Citrulline is synthesized in mitochondria of Neurospora crassa from ornithine and carbamoyl phosphate. In mycelia grown in minimal medium, carbamoyl phosphate limits citrulline (and arginine) synthesis. Addition of arginine to such cultures reduces the availability of intramitochondrial ornithine, and ornithine then limits citrulline synthesis. We have found that for some time after addition of excess arginine, carbamoyl phosphate synthesis continued. Very little of this carbamoyl phosphate escaped the mitochondrion to be used in the pyrimidine pathway in the nucleus. Instead, mitochondrial carbamoyl phosphate accumulated over 40-fold and turned over rapidly. This was true in ornithine- or ornithine carbamoyltransferase-deficient mutants and in normal mycelia during feedback inhibition of ornithine synthesis. The data suggest that the rate of carbamoyl phosphate synthesis is dependent to a large extent upon the specific activity of the slowly and incompletely repressible synthetic enzyme, carbamoyl-phosphate synthetase A. In keeping with this conclusion, we found that when carbamoyl-phosphate synthetase A was repressed 2-10-fold by growth of mycelia in arginine, carbamoyl phosphate was still synthesized in excess of that used for residual citrulline synthesis. Again, only a small fraction of the excess carbamoyl phosphate could be accounted for by diversion to the pyrimidine pathway. The continued synthesis and turnover of carbamoyl phosphate in mitochondria of arginine-grown cells may allow rapid resumption of citrulline formation after external arginine disappears and no longer exerts negative control on ornithine biosynthesis.     

Oxidative inactivation of carbamoyl phosphate synthetase (ammonia). Mechanism and sites of oxidation, degradation of the oxidized enzyme, and inactivation by glycerol, EDTA, and thiol protecting agents.

Acetylglutamate and ATP accelerate the oxidative inactivation of carbamoyl phosphate synthetase I by mixtures of Fe3+, ascorbate, and O2, but the mechanism of the inactivation differs with each ligand. In the presence of acetylglutamate, MgATP prevents, Mg2+, Mn2+, and catalase have no effect, and EDTA increases the inactivation, and the two phosphorylation steps of the enzyme reaction are lost simultaneously. The inactivation appears to be mediated by dehydroascorbate and is associated with the reversible oxidation of the highly reactive cysteines 1327 and 1337 and with oxidation of non-thiolic groups in the second 40-kDa domain (the enzyme consists of 4 domains of 40, 40, 60, and 20 kDa, from the amino terminus). The data are consistent with oxidation of groups at or near the site for ATPA (ATPA yields Pi; ATPB yields carbamoyl phosphate), and with the location of this site at the interphase between the second 40-kDa and the COOH-terminal domains. The oxidative inactivation promoted by ATP is inhibited by Mg2+, Mn2+, catalase, and EDTA, is not mediated by dehydroascorbate, and is not associated with oxidation of cysteines 1327 and 1337. Groups in the 60-kDa domain are oxidized. The phosphorylation step involving ATPB is lost preferentially, and the inactivation and the binding of ATPB exhibit the same dependency on the concentration of ATP. The results indicate that the oxidation is catalyzed by FeATP bound at the site for ATPB and support the binding of ATPB in the 60-kDa domain. We also demonstrate that mercaptoethanol, reducing impurities in glycerol, and dithioerythritol, in the presence of EDTA, replace ascorbate in the oxidative system. In addition, we study the influence of the oxidation on the degradation of the enzyme by rat liver lysosomes, mitochondria, and cytosol.     

The stimulation of the mitochondrial respiration by citrulline synthesis.

1. The influence of ammonia and ornithine on the oxygen uptake and the formation of citrulline was investigated with isolated rat liver mitochondria. The experiments were performed in a cytosol-like saline medium at 38 degrees C. 2. Under these conditions an increase of the respiration rate by ammonia and ornithine was observed, but a small response to external ADP, only. The missing stimulation by ADP was due to a partial inhibition of the respiratory chain by traces of zinc (approximately 1 microM) present in the medium. This inhibition was only detected at low concentrations of mitochondria. 3. For activation of respiration by ammonia plus ornithine two different processes were responsible: (i) chelation of the inhibiting zinc by ornithine, which could be prevented by EDTA; (ii) ADP production in the matrix space during formation of carbamoyl phosphate, which could be prevented by oligomycin but not by carboxyatractyloside. 4. This stimulus of the carbamoyl phosphate formation and of the equivalent citrulline synthesis on the mitochondrial respiration ran to 12% of that increase caused by phosphorylation of external ADP. The maximum rate of citrulline formation was limited by the activity of carbamoyl phosphate synthetase. 5. Added ADP suppresses the production of citrulline probably by the exchange of extramitochondrial ADP versus intramitochondrial ATP. The data suggest a common adenine nucleotide pool delivering ATP to the adenine nucleotide translocase as well as to the carbamoyl phosphate synthetase.     

Stimulatory effect of arginine on acetylglutamate synthesis in isolated mitochondria of mouse and rat liver.

N-Acetyl-L-glutamate synthetase (EC 2.3.1.1) catalyses the synthesis of N-acetyl-L-glutamate, an allosteric activator of carbamoyl-phosphate synthetase I in the liver of ureotelic animals, and the first enzyme is activated specifically by arginine. We have proposed that arginine can stimulate acetylglutamine synthetase in vivo and thereby increase the mitochondrial content of acetylglutamate. The effects of arginine on acetylglutamate synthesis in isolated mitochondria were investigated in detail in the present work. When rat liver mitochondria were isolated and incubated with [14C]glutamate and unlabelled acetate as substrates, acetyl[14C]glutamate synthesis in the mitochondria was more extensive in the presence than in the absence of L-arginine. There was no significant difference between the specific radioactivities of intramitochondrial [14C]glutamate in the presence and absence of arginine. When rat liver mitochondria were incubated with [14C]acetate and unlabelled glutamate as substrates, arginine also stimulated acetyl[14C]glutamate synthesis in the isolated mitochondria. L-Lysine or L-homoarginine, which does not activate acetylglutamate synthetase, had no effect on acetylglutamate synthesis, in the isolated mitochondria. The arginine concentration giving half-maximal synthesis of acetylglutamate in isolated mitochondria was about 50 microM, which is in the range of physiological concentrations of arginine in the liver. As we previously reported [Kawamoto, Ishida, Mori & Tatibana (1982) Eur. J. Biochem. 123, 637-641], the sensitivity of acetylglutamate synthetase to arginine activation undergoes marked changes after food ingestion. The extent of arginine activation of acetylglutamate synthesis in isolated mitochondria correlated well with the sensitivity of acetylglutamate synthetase extracted from the mitochondria to arginine activation. These data lend further support to the idea that arginine itself activates the mitochondrial synthesis of acetylglutamate.     

Citrulline synthesis in rat tissues and liver content of carbamoyl phosphate and ornithine

Rat liver ornithine carbamoyltransferase appears to be located exclusively in the mitochondria; the activity that is found in the soluble fraction is indistinguishable from mitochondrial ornithine carbamoyltransferase by simple kinetic criteria, and seems to result from breakage of mitochondria during homogenization. Of several rat tissues studied, only the liver and the mucosa of small intestine contain significant amounts of ornithine carbamoyltransferase; the activity in intestinal mucosa is less than one thousandth of that in liver. Qualitatively, this distribution coincides with that of carbamoyl phosphate synthetase I and its cofactor, acetylglutamate. The rat liver contents of carbamoyl phosphate and ornithine were 0.1 and 0.15mumol/g wet wt. of tissue respectively. On the basis of these values, it is proposed that in vivo the ornithine carbamoyltransferase activity of liver may be much lower than its maximal activity in vitro might suggest.     

Purification and properties of ornithine carbamoyl transferase from liver of Squalus acanthias

Citrulline synthesis from ammonia by hepatic mitochondria in elasmobranchs involves intermediate formation of glutamine as the result of the presence of high levels of glutamine synthetase and a unique glutamine- and N-acetyl-glutamate-dependent carbamoyl phosphate synthetase, both of which have properties unique to the function of glutamine-dependent synthesis of urea, which is retained in the tissues of elasmobranchs at high concentrations for the purpose of osmoregulation [P.M. Anderson and C.A. Casey (1984) J. Biol. Chem. 259, 456-462; R.A. Shankar and P.M. Anderson (1985) Arch. Biochem. Biophys. 239, 248-259]. The objective of this study was to determine if ornithine carbamoyl transferase, which catalyzes the last step of mitochondrial citrulline synthesis and which has not been previously isolated from any species of fish, also has properties uniquely related to this function. Ornithine carbamoyl transferase was highly purified from isolated liver mitochondria of Squalus acanthias, a representative elasmobranch. The purified enzyme is a trimer with a subunit molecular weight of 38,000 and a native molecular weight of about 114,000. The effect of pH is significantly influenced by ornithine concentration; optimal activity is at pH 7.8 when ornithine is saturating. The apparent Km values for ornithine and carbamoyl phosphate at pH 7.8 are 0.71 and 0.05 mM, respectively. Ornithine displays considerable substrate inhibition above pH 7.8. The activity is not significantly affected by physiological concentrations of the osmolyte urea or trimethylamine-N-oxide or by a number of other metabolites. The results of kinetic studies are consistent with a steady-state ordered addition of substrates (carbamoyl phosphate binding first) and rapid equilibrium random release of products. Except for an unusually low specific activity, the properties of the purified elasmobranch enzyme are similar to the properties of ornithine carbamoyl transferase from mammalian ureotelic and other species and do not appear to be unique to its role in glutamine-dependent synthesis of urea for the purpose of osmoregulation.