Competition between extramitochondrial and intramitochondrial ATP-consuming processes.

The relationship between intra- and extramitochondrial ATP utilization was investigated in liver mitochondria isolated from normally fed, starved and high-protein fed rats. ATP export was provoked by adding a hexokinase-glucose-trap and intramitochondrial ATP consumption by adding ammonia, bicarbonate and ornithine in order to stimulate citrulline synthesis. Both processes compete for ATP produced via oxidative phosphorylation; the rate of citrulline formation declines as the extramitochondrial [ATP]/[ADP] ratio decreases. It is concluded that ATP for adenine nucleotide translocation and that for carbamoyl phosphate synthesis are delivered from a common intramitochondrial pool of adenine nucleotides. In mitochondria from rats with a high-protein diet, citrulline synthesis greatly stimulates the rate of oxidative phosphorylation (about two thirds of state 3 respiration). Under these conditions the intramitochondrial [ATP]/[ADP] ratio is significantly reduced. The intramitochondrial [ATP]/[ADP] ratio is not in thermodynamic equilibrium with the extramitochondrial one.     

Partial characterization of ornithine carbamoyltransferase in three microalgae : anabolic role only

Although the existence of isozymes of ornithine carbamoyltransferase (carbamoylphosphate:l-ornithine carbamoyltransferase, EC 2.1.3.3) in higher plants has been reported, and the possibility exists that one or more of these operates catabolically to produce ornithine and carbamoylphosphate from citrulline and inorganic phosphate, no proof has been forthcoming. In view of the fact that many unicellular algae degrade arginine via arginine deiminase to citrulline and ammonium, and that the pathway of utilization of citrulline is unknown, we decided to investigate the possibility of the presence of a catabolic form of ornithine carbamoyltransferase in three microalgae known to have arginine deiminase activity. These were Chlorella autotrophica, Chlorella saccharophila, and Dunaliella tertiolecta. Our results show that the properties of OCT from these three algae are similar to OCTs from many higher plants with respect to general kinetics (K_m values for ornithine and carbamoylphosphate), substrate inhibition by ornithine at high pHs, apparent sequential ordered kinetic mechanisms and paucity of apparent regulatory properties. Our data indicate an exclusively anabolic role of ornithine carbamoyltransferase in these algae.     

Developmental changes of citrullinogenesis, mitochondrial N-acetylglutamate content and N-acetylglutamate synthetase in fetal and neonatal rats

A low citrullinogenesis (less than 60 per cent of the adult value) was observed throughout the suckling period when mitochondria isolated from newborn rat liver were incubated in vitro with L-glutamate or succinate as oxidizable substrates. The adult value was reached after weaning. From birth to weaning, intact mitochondria synthesized more citrulline when supplemented with L-glutamate than with succinate. The low citrullinogenesis could not be explained by low carbamoylphosphate synthetase-I and ornithine transcarbamoylase activities that reached adult values at birth. The decreased citrullinogenesis seen for the first three days of life seemed to be related to the low intramitochondrial concentration of N-acetylglutamate, an activator of the carbamoylphosphate synthetase-I. The concentration of this activator did not differ from that reported for adult rat liver mitochondria after the fourth day of life. The discrepancy between the normal value of N-acetylglutamate concentration and the low activity of the N-acetylglutamate synthetase (15 to 30 per cent of the adult activity) is discussed on the basis of acetyl-CoA or L-glutamate availability in mitochondria isolated from newborn or young rats.     

Arginine uptake by isolated rat liver mitochondria

The question of arginine uptake by mitochondria is important in that arginine is an allosteric effector of N-acetylglutamate synthetase. Thus, changes in mitochondrial arginine concentration have the potential for acutely modifying levels of N-acetylglutamate, a compound necessary for maximal activity of carbamyl phosphate synthesis. Mitochondria were isolated from chow-fed rats, incubated with [guanido-¹⁴C]arginine and were centrifuged through silicon oil into perchloric acid for determination of intramitochondrial metabolites. Arginine was separated from urea by cation-exchange resin. Mitochondrial water space was determined by [¹⁴C]urea arising from arginase activity associated with the mitochondrial preparations. Extramatrix space was determined by parallel incubations with [inulin-¹⁴C]carboxylic acid or [¹⁴C]sucrose There was considerable degradation of arginine by arginase associated with the mitochondrial preparation. This was inhibited by 7 mM ornithine and 7 mM lysine. Arginine was concentrated intramitochondrially to 4-times the extramitochondrial levels. The concentration ratio was decreased in the presence of ornithine and lysine but not with citrulline, NH₄Cl, glutamate, glutamate or leucine. No uptake was observed when mitochondria were incubated at 0°C. Mitochondria did not concentrate citrulline.     

Relations between extramitochondrial and intramitochondrial adenine nucleotide systems

The control of oxidative phosphorylation by the extramitochondrial $\text{[}\text{ATP}\text{]}\text{[}\text{ADP}\text{]}$ ratio and [P_i] was investigated by incubations of isolated mitochondria with an ADP regenerating system and by a new perifusion technique using glass filters for immobilization of mitochondria. With mitochondria from different sources oxidizing different substrates and with both techniques, similar results were obtained. Changes of the extramitochondrial $\text{[}\text{ATP}\text{]}\text{[}\text{ADP}\text{]}$ ratio from about 100 to 5 transfer mitochondria from the resting state (state 4) to the fully active state (state 3). The importance of the adenine nucleotide translocator in this transition was demonstrated by the influence of its specific inhibitor carboxyatractyloside. The sensitivity to the inhibitor was more pronounced in states with high $\text{[}\text{ATP}\text{]}\text{[}\text{ADP}\text{]}$ ratios than in the fully active state. In the hexokinase-glucose system the action of the inhibitor caused a transition to a new steady state, where a decreased $\text{[}\text{ATP}\text{]}\text{[}\text{ADP}\text{]}$ ratio overcomes the inhibition. Thus, a partial inhibition of the translocator shifted the control characteristics to lower $\text{[}\text{ATP}\text{]}\text{[}\text{ADP}\text{]}$ ratios. When the concentration of inorganic phosphate was decreased, the main effect was a reduction of the maximum rate of oxidative phosphorylation (i.e., in state 3), whereas the $\text{[}\text{ATP}\text{]}\text{[}\text{ADP}\text{]}$ sensitive range was not altered. This effect is caused by changes in the intramitochondrial phosphorylation potential. Furthermore, this indicates that the kinetic properties of the adenine nucleotide translocator prevent a simple equilibration of the phosphorylation potential across the inner membrane. This is also demonstrated by the fact that the extramitochondrial formation of glucose-6-phosphate and the intramitochondrial synthesis of citrulline compete for ATP.     

Orotic acid biosynthesis in rat liver: studies on the source of carbamoylphosphate.

Measurements of the incorporation of radiolabeled precursors into orotic acid in tissue slices and minces provided evidence of the participation of the intramitochondrial carbamoylphosphate synthetase (CPSase-I) in the de novo biosynthesis of pyrimidines in rat liver. Ammonia, the only nitrogen source utilized by CPSase-I, markedly stimulated the incorporation of NaH¹⁴CO₃ into orotic acid in liver slices, and ornithine, which enhances the intramitochondrial consumption of carbamoylphosphate (CP) in citrulline synthesis, antagonized the stimulation by ammonia. Sensitivity of the incorporation of NaH¹⁴CO₃ into orotic acid to stimulation by ammonia was found to increase with age in concert with the emergence of CPSase-I in the liver during late fetal and neonatal development. Tissues lacking in CPSase-I activity did not exhibit the responses to ammonia and ornithine observed with the adult rat liver. While the occurrence of CPSase-I in the liver contributes extensively toward the exceptionally high capacity of that tissue for the de novo biosynthesis of orotic acid, our results also indicate that the physiological rate of orotic acid biosynthesis in rat liver is approximately one-third of capacity; the incorporation of NaH¹⁴CO₃ into orotic acid averaged 488 nmol/g of tissue in 3 h in the presence of toxic levels of ammonia, but declined to 160 nmol/g of tissue in 3 h when physiological levels of both ammonia and ornithine were provided. However, the rate of orotic acid biosynthesis observed with physiological concentrations of ammonia and ornithine could be reduced further, to about one-quarter of the physiological rate, by providing additional ornithine; thus, physiological levels of ornithine do not prevent the escape of intramitochondrial CP into the cytoplasm. Finally, over 80% of the incorporation of NaH¹⁴CO₃ into orotic acid at physiological levels of ammonia and ornithine was found to be ammonia dependent, and all but a small fraction of the ammonia-dependent incorporation could be blocked by providing ornithine in amounts in excess of physiological. These results indicate that CPSase-I is the major source of CP in the biosynthesis of hepatic pyrimidines under normal (physiological) conditions as well as in ammonia toxicity.     

Nδ-(phosphonacetyl)-L-ornithine,a potent transition state analogue inhibitor of ornithine carbamoyltransferase

$\text{N}$^δ-(Phosphonacetyl)-L-ornithine, a transition state analogue for the reaction catalyzed by ornithine carbamoyltransferase (EC 2.1.3.3), was synthesized. It strongly inhibited bovine liver ornithine carbamoyltransferase. The inhibition was competitive with respect to carbamoyl-phosphate; the apparent $\text{K}$_m values for carbamoyl-phosphate were 15 μM in 0.05 M $\text{N}$-2-hydroxyethylpiperazine-$\text{N′}$-2-ethanesulfonate (pH 7.2) and 33 μM in 0.1 M Tris-HCl (pH 8.5), and the inhibition constants at pH 7.2 and 8.5 were 7.1 and 4.7 nM, respectively. The inhibition was non-competitive with L-ornithine, the other substrate of the enzyme. This analogue may provide an effective reagent for the elucidation of carbamoyl-phosphate metabolism and its regulation in the liver of ureotelic animals.     

The localization within plant cells of enzymes involved in arginine biosynthesis

Studies were carried out to determine the distribution of the following: (1) carbamoyl phosphate synthetase (EC 2.7.2.9), (2) ornithine carbamoyltransferase (EC 2.1.3.3), (3) argininosuccinate synthetase (EC 6.3.4.5), and (4) argininosuccinate lyase (EC 4.3.2.1) in soybean cells grown in suspension culture. Protoplasts were produced from the soybean cells by treatment with cellulase (EC 3.2.1.4) and pectinase (EC 3.2.1.15); the protoplasts were then ruptured by osmotic shock with distilled water. This treatment was followed by differential centrifugation and sucrose density gradient centrifugation to isolate various organelle fractions including mitochondria and plastids. Examination of these fractions using specific enzyme assays showed that carbamoylphosphate synthetase and ornithine carbamoyltransferase were localized in a fraction found to be composed primarily of plastids. Argininosuccinate synthetase and argininosuccinate lyase appeared to be associated with either the cytosol or a membrane fraction in close association with the cytosol such as the endoplasmic reticulum or protoplast membrane.     

Intramitochondrial localisation of glycine decarboxylase in spinach leaves

Intact spinach leaf mitochondria are capable of oxidising glycine with good respiratory control and the oxidation is coupled to 3 phosphorylation sites. The intramitochondrial localisation of glycine decarboxyllation has been studied and it is demonstrated that the enzyme system is associated with the inner membrane of spinach leaf mitochondria. Both glycine decarboxylation and glycine dependent O₂ uptake are stimulated by ADP and FCCP and are sensitive to electron transport inhibitors. Both processes showed no requirements for co-factors. We suggest that glycine decarboxylase is coupled to the electron transport chain $\text{via}$ an NAD⁺-linked system and that during rapid photorespiration glycine oxidation synthesises considerable amounts of ATP outside of the chloroplast.     

Functional efficiency of mitochondrial membrane of rats with hepatic chronic iron overload

The effect of $\text{in}\text{vivo}$ hepatic iron overload, induced by two different amounts of iron, on the energy-transducing efficiency of the mitochondrial membrane has been examined. It has been found that when the epatic iron concentration is up to a threshold value mitochondria present an anomalous membrane potential. Addition of oligomycin fully restitutes it. A low content of intramitochondrial K⁺ is connected with this pathological condition. A relative lack of antioxidant capability is parallely exhibited by these mitochondria. A possible involvement of lipid peroxidation process $\text{in}\text{vivo}$ in causing the membrane potential drop and the net efflux of intramitochondrial K⁺ is suggested.     

The hormonal stimulation of ureogenesis in isolated hepatocytes through increases in mitochondrial ATP production

Isolated hepatocytes incubated with 2 mm ornithine-10 mm glutamine as substrates and challenged with either glucagon, epinephrine, or phenylephrine exhibited stimulated rates of urea production, and mitochondria isolated from these cells displayed an increased rate of energy-dependent citrulline formation. There was no change in the total carbamyl phosphate synthetase I activity, nor mitochondrial content of the positive effector N-acetyl glutamate after acute hormonal treatment. The time of onset of ureogenesis and its sensitivity to glucagon were compared with stimulation of glucose production from lactate-pyruvate. No apparent differences in time of onset or sensitivity of the responses were observed indicating both pathways may be stimulated by a common mechanism. Mitochondria prepared from cells treated with catecholamines exhibited increased rates of State 3 respiration and increased uncoupler-dependent ATPase activity, in addition to the increased rates of citrulline formation. There was also an elevated intramitochondrial content of ATP and an increased $\text{ATP}\text{ADP}$ ratio. The catecholamine-induced stimulation of ureogenesis was mediated by an α-adrenergic cyclic AMP independent mechanism. The addition of the α-adrenergic antagonist, dihydroergotamine, blocked both the epinephrine-induced stimulation of ureogenesis and also the stimulated functions in the isolated mitochondria. dl-Propranolol, a β-antagonist, inhibited the rise in cyclic AMP due to epinephrine, but had no effect on any of the other reactions measured. The effects of catecholamines on citrulline formation and urea production are correlated with the increased capacity of the mitochondria to generate ATP. It is suggested that both glucagon and catecholamines, acting via independent mechanisms, stimulate electron transport and the activity of the ATP-forming enzyme complex. The consequent elevated intramitochondrial ATP levels and $\text{ATP}\text{ADP}$ ratio enhance the rate of citrulline formation and hence ureogenesis.     

Arginine metabolism in Saccharomyces cerevisiae: subcellular localization of the enzymes.

Subcellular localization of enzymes of arginine metabolism in Saccharomyces cerevisiae was studied by partial fractionation and stepwise homogenization of spheroplast lysates. These enzymes could clearly be divided into two groups. The first group comprised the five enzymes of the acetylated compound cycle, i.e., acetylglutamate synthase, acetylglutamate kinase, acetylglutamyl-phosphate reductase, acetylornithine aminotransferase, and acetylornithine-glutamate acetyltransferase. These enzymes were exclusively particulate. Comparison with citrate synthase and cytochrome oxidase, and results from isopycnic gradient analysis, suggested that these enzymes were associated with the mitochondria. By contrast, enzymatic activities going from ornithine to arginine, i.e., arginine pathway-specific carbamoylphosphate synthetase, ornithine carbamoyltransferase, argininosuccinate synthetase, and argininosuccinate lyase, and the two first catabolic enzymes, arginase and ornithine aminotransferase, were in the "soluble" fraction of the cell.     

A re-evaluation of conditions required for an accurate estimation of the extramitochondrial ATPADP ratio in isolated rat-liver mitochondria

The values reported in the literature for the extramitochondrial $\text{ATP}\text{ADP}$ ratio in resting rat-liver mitochondria (State 4) vary widely. The conditions required for an accurate determination of this parameter were therefore investigated. (1) In experiments with rat-liver mitochondria incubated under State-4 conditions, it was found that the extramitochondrial $\text{ATP}\text{ADP}$ ratio, as calculated from the values measured in neutralised perchloric acid extracts, was lower than that estimated from the concentrations of creatine and creatine phosphate, using the metabolite indicator method. The discrepancy is due to hydrolysis of ATP occurring in the presence of perchloric acid. (2) Conditions are described for minimising ATP hydrolysis in the presence of perchloric acid, and include the use of low concentrations of perchloric acid, short times of exposure to the acid before neutralisation, low temperatures and the presence of excess EDTA. Under these conditions, the values obtained for the extramitochondrial $\text{ATP}\text{ADP}$ ratio agreed with those calculated by the metabolite indicator method, provided ratios do not exceed the value of 100. (3) In cases where the extramitochondrial $\text{ATP}\text{ADP}$ does exceed 100, phenol/chloroform/isoamyl alcohol must be used to quench the reactions, as described by Slater et al. (Slater, E.C., Rosing, J. and Mol, A. (1973) Biochim. Biophys. Acta 292, 534–553). With this method, the extramitochondrial $\text{ATP}\text{ADP}$ ratio was found to have a value of more than 1000 in rat-liver mitochondria incubated with succinate + rotenone in the resting state (pH 7.0; T = 37°C), in agreement with Slater et al.     

Altered restriction of nuclear RNA during incubation in vitro

Nuclei were isolated from rat liver and incubated $\text{in}\text{vitro}$ in two commonly employed RNA transport assays. Released [¹⁴C] RNA was isolated and hybridized with filter-bound DNA in the presence of competing cytoplasmic RNA. A significant portion of RNA which was transported to either medium was not represented in cytoplasmic RNA. These results indicate that the restriction of some sequences to the nucleus $\text{in}\text{vivo}$ is not maintained $\text{in}\text{vitro}$.     

Complementary expression of glutamine synthetase and carbamoylphosphate synthetase I in ornithine carbamoyltransferase-deficient mouse liver (spf–ash mouse)

 Glutamine synthetase and carbamoylphosphate synthetase I expression was examined immunohistochemically in livers of spf–ash homozygous and hemizygous mice, in which one of the urea cycle enzymes (ornithine carbamoyltransferase) is deficient and hyperammonemic disorders are obvious. In the mutant adult mouse liver, only hepatocytes lining central veins expressed glutamine synthetase. In contrast, other hepatocytes expressed carbamoylphosphate synthetase I but not glutamine synthetase. This complementary expression pattern is similar to that seen in wild-type mouse liver. In the liver of mutant young mice, which showed severe retarded growth and abnormal hair and skin development, the developmental expression pattern of both enzymes was also similar to that of the corresponding wild-type liver. However, suppression of carbamoylphosphate synthetase I expression in the pericentral hepatocytes occurred later in the mutant than in wild-type liver. These results show that high plasma concentrations of ammonium ions, which are one of the substrates for both the enzymes, do not change their complementary expression. Instead they support the idea that factor(s) associated with central veins rather than humoral factors direct pericentral hepatocytes to express glutamine synthetase and to suppress carbamoylphosphate synthetase I expression. Accepted: 24 April 1997     

Mitochondrial compartmentation of metabolic CO2 resulting from its site of origin in relation to urea synthesis

In isolated hepatocytes the entry into urea of metabolic ¹⁴CO₂; derived from [¹⁴C] formate is modified by the addition of dichloroacetate and hydroxypyruvate. An explanation is that this results from changes in the cytoplasmic/mitochondrial pH gradient. ¹⁴CO₂, derived from [1-¹⁴C]alanine enters into urea more readily than ¹⁴CO₂ arising from [1-¹⁴C]glutamate. It is proposed that the difference, which is more than 4-fold, is indicative of a preferred pathway for metabolic CO₂ in liver mitochondria from pyruvate dehydrogenase to carbamoylphosphate synthetase than form oxoglutarate dehydrogenase. Acetazolamide inhibition of carbonic anhydrase is without effect on this observed incorporation into urea.     

The effects of glucagon and epinephrine on two preparations of cardiac mitochondria

The effects of $\text{in}$$\text{vivo}$ administration in epinephrine on calcium uptake were measured in two preparations of heart mitochondria, intermyofibrillar (IMF) and subsarcolemmal (SSL) using either ⁴⁵Ca²⁺ or murexide to follow calcium movement. The administration of either hormones resulted in an increased calcium uptake in both preparation of mitochondria subsequently isolated. This increase might be the consequence of the increased State 3 respiration, also evoked by hormones. The possibility is raised that the inotropic actions of glucagon and epinephrine might be partially mediated by mitochondria.     

The duplication of arginine catabolism and the meaning of the two ornithine carbamoyltransferases in Bacillus licheniformis.

Arginine deiminase and carbamate kinase activities are shown to be present in cell-free extracts of $\text{Bacillus licheniformis}$. This gives the rationale for the occurrence of an arginine-inducible ornithine carbamoyltransferase in this organism and suggests that, $\text{in vivo}$ and under the proper conditions, this enzyme is able to catalyze the phosphorolysis of citrulline. This also shows that this Bacillus species has two arginine catabolic pathways. This duplication appears to be under the control of O₂.     

Measurement of the intramitochondrial P/O ratio

Measurements of the initial rate of ATP synthesis and the initial rate of oxygen consumption in mitochondria in which transport of ADP, Pi and ATP were inhibited were used to obtain a value for the intramitochondrial $\text{P}\text{O}$ ratio. With succinate as substrate this method yielded a $\text{P}\text{O}$ ratio of 2.8 for the phosphorylation of intramitochondrial ADP.