Aroclor 1254 treatment and fasting influences on rat liver mitochondrial carbamoyl phosphate synthesis with ADP and ATP.

Previous studies have shown that the polychlorinated biphenyl mixture, Aroclor 1254 (ARO), -induced wasting in male rats is associated with increased permeability of hepatic mitochondria. This was correlated with hyperuremia and stimulated urea synthesis, hypoglycemia and suppressed glucogenesis after an ammonium acetate injection, and decreased retention of assimilated nitrogen and food intake. For ARO-toxic rats (100 mg/kg, ip, for 1, 2, and 4 days) versus Tween 80-treated, ad libitum-fed controls, mitochondrial carbamoyl phosphate (CP) formation (the initial step in urea synthesis from NH4+) was progressively stimulated for the duration of treatment from NH4+ and ATP but not from NH4+ and ADP. ARO maximal stimulation of CP formation also correlated with significant loss in body weight. Mitochondrial ornithine transcarbamoylase synthesis of citrulline from ornithine and carbamoyl phosphate was also stimulated. In comparison to fasted rats (24 hr), mitochondrial CP synthesis from NH4+ was enhanced with ADP but not with ATP. This ARO uncoupling of mitochondrial NH4+ metabolism and stimulation of CP formation with exogenous ATP and citrulline synthesis may have resulted from increased availability of substrates and cofactors in the matrix space, leakage of enzymes from the matrix, or a combination of these effects. These results are consistent with an increased inner membrane permeability and fragility during isolation and assays. In agreement with our previous studies, the data show that ARO exposure poises hepatic mitochondria toward the synthesis of urea intermediates.

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.

In vivo synthesis of carbamyl phosphate from NH3 by the large subunit of Escherichia coli carbamyl phosphate synthetase.

The cloned carAB operon of Escherichia coli coding for the small and large subunits of carbamyl phosphate synthetase has been used to construct a recombinant plasmid with a 4.16 kilobase ClaI fragment of the car operon that lacks the major promoters, P1 and P2. The plasmid, pHN12, carries a functional carB gene. A mutant E. coli strain lacking both subunits of carbamyl phosphate synthetase when transformed with pHN12 overproduces the large subunit by 200-fold (8-10% of the cellular protein). The elevated levels of the large subunit enable the transformed cells to utilize NH3 but not glutamine as nitrogen donor for carbamyl phosphate synthesis. The large subunit has been purified from the overexpressing strain. The purified native large subunit is capable of synthesizing carbamyl phosphate from ammonia, HCO-3, and ATP. The kinetic properties of the large subunit compared with the holoenzyme indicate that the Michaelis constants of the large subunit for HCO-3 and ATP are modulated by its association with the small glutamine binding subunit.

Enzymes of pyrimidine biosynthesis in Crithidia luciliae.

Evidence has been obtained for the presence of enzymes of both the de novo and salvage pyrimidine pathways in the protozoan parasite, Crithidia luciliae. Carbamyl phosphate synthetase-II activity could not be unequivocally demonstrated in crude extracts. However, a distinct peak of activity with a molecular weight of approximately 500 000 was observed following chromatography on Sepharose CL-6B. The enzyme preferentially utilised glutamine with respect to ammonia. It was inhibited by UTP and 5-phosphoribosyl-1-diphosphate had a small activating effect. Carbamyl phosphate synthesis by a 'phosphorolytic' citrullinase could not be demonstrated. The ensuing three de novo enzymes could also be separated on Sepharose CL-6B. Approximate molecular weights were estimated: aspartate transcarbamylase (150,000); dihydroorotase (90,000) and dihydroorotate dehydrogenase (70,000). As reported previously, orotate phosphoribosyltransferase and orotidylate decarboxylase were particulate, being associated with the glucosome. Activities of the salvage enzymes, uracil phosphoribosyltransferase, uridine phosphorylase and uridine nucleosidase were observed. All enzymes were cytoplasmic. No uridine kinase activity was detected.

Does ornithine stimulate carbamoylphosphate synthetase?

The effect of ornithine on carbamoylphosphate formation of rat liver mitochondria treated with Triton X 100 was studied. The rate of carbamoylphosphate accumulation and citrulline formation depended on the ATP-, Pi-, N-acetylglutamate and protein concentration. At optimal conditions the rate of citrulline formation was at least 1.5-fold higher than the rate at which carbamoylphosphate accumulated (ornithine absent). A significant correlation between the amount of carbamoylphosphate formed and the citrulline/carbamoylphosphate ratio (ornithine effect) was found. In mitochondria the presence of a carbamoylphosphate degrading enzyme could be demonstrated. This enzyme may be one factor for the differences in the rate of carbamoylphosphate accumulation and the rate of citrulline synthesis.

[Determination of the carbamate kinase activity of bacteria]. / Opredelenie karbamatkinaznoi aktivnosti bakterii.

The activity of carbamate kinase (EC 2.5.2.2) was determined in bacteria using a simple modified procedure. Carbamoyl phosphate produced under the action of carbamate kinase carbamoylated ammonia in a reaction which was not enzyme-catalyzed yielding urea that was assayed by the colorimetric technique. The activity of carbamate kinase was found by this method in a number of microorganisms. The method can be used to study other enzymes synthesizing carbamoyl phosphate. The advantages of the method over other techniques are discussed.

[Carbamoyl phosphate biosynthesis in Streptococci]. / O biosinteze karbamoilfosfata u streptokokkov.

Nine streptococcal strains belonging to different serological groups (A, B, C, D) were shown to synthesize carbamoyl phosphate from ammonium hydrocarbonate and ATP. The reaction was catalyzed by carbamate kinase (EC 2.7.2.2). The speed of the reaction was evaluated according to the increase of the content of citrullin (the combination of carbamate kinase and ornithine transcarbamoylase). The representatives of different serological groups were found to have quantitative differences in carbamate kinase activity: the highest specific activity (13 nmol of citrullin per minute in 1 mg of dried microbial biomass) was detected in group A streptococci, while group D streptococci showed the lowest specific activity (0.5 nmol).

[Carbamyl phosphate and citrulline formation by phototrophic bacteria]. / Obrazovanie karbamoilfosfata i tsirullina fototrofnymi bakteriiami.

The purple bacteria Ectothiorhodospira shaposhnikovii and Rhodospirillum rubrum are capable of synthesizing citrulline in the presence of ammonium hydrocarbonate, ornithine, ATP and Mg2+ ions. Citrulline biosynthesis by these phototrophic bacteria is presumed to be catalysed by carbamate kinase and ornithine transcarbamoylase. Therefore, certain phototrophic bacteria can assimilate carbon dioxide and ammonia for biosynthesis of amino acids, in particular, citrulline and, apparently, arginine.

Regulation of carbamylphosphate synthesis in Serratia marcescens.

Serratia marcescens HY possessed a single carbamylphosphate synthase (CPSase) which was subject to cumulative repression by arginine and a pyrimidine. CPSase did not appear to be a part of a multifunctional enzyme complex as is the case for other enzymes of pyrimidine biosynthesis in this organism. CPSase was purified to homogeneity. The molecular weight of the enzyme was estimated to be 167,000 by sucrose density gradient ultracentrifugation. The double-reciprocal plot for magnesium adenosine triphosphate was linear, yielding a Km value of 2.5 mM. The enzyme utilized either glutamine (Km, 0.1 mM) or NH3 (Km, 10.5 mM) as a nitrogen donor in the reaction. CPSase activity was subject to activation by ornithine and feedback inhibition by uridine monophosphate, as is the case for other enteric bacteria. Carbamate kinase activity, detected in crude extracts of S. marcescens, was shown to be due to a constitutive acetate kinase. The absence of carbamate kinase from S. marcescens HY is consistent with the inability of this organism to utilize arginine as a source of energy under anaerobic conditions.

Sources of ammonia for mammalian urea synthesis.

The initial rate of incorporation of [15N]alanine into the 6-amino group of the adenine nucleotides in rat hepatocytes was about one-eighteenth of the rate of incorporation into urea. Thus the purine nucleotide cycle cannot provide most of the ammonia needed in urea synthesis for the carbamoyl phosphate synthase reaction (EC 2.7.2.5). On the other hand, contrary to the view expressed by McGivan & Chappell [(1975) FEBS Lett. 52, 1--7], the experiments support the view that hepatic glutamate dehydrogenase can supply the required ammonia.

Carbamyl phosphate synthesis in Bacillus subtilis.

In vitro and "in situ" assays have been developed to test the carbamyl phosphate synthetase (CPSase) activity of a series of pyrimidine-requiring mutants of Bacillus subtilis. The enzyme has been shown to be highly unstable, and was successfully extracted only in the presence of 10% glycerol and 1 mM dithiothreitol (Cleland's reagent). It loses activity rapidly when sonicated or when treated with lysozyme. Genetic studies, using mutants, indicate that B. subtilis may possess two CPSases. This possibility and its physiological consequences were probed enzymatically. CPSase activity has been shown to undergo inhibition by both uridine triphosphate and dihydroorotate; activation has been demonstrated in response to phosphoribosyl pyrophosphate (PRPP) and (to a lesser extent) ornithine.