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The conflicting data on the binding of the two molecules of ATP that are involved in the overall reaction catalyzed by carbamoyl-phosphate synthetase (CPS) of Escherichia coli, and a mechanism recently proposed for this reaction, has led us to reexamine ATP binding using pulse/chase techniques. With [gamma-32P]ATP and bicarbonate in the pulse solution, there is a positive intercept at zero time of approximately 1 mol Pi/mol CPS in the plot of 32Pi formation against time, irrespective of whether the incubation is terminated by the addition of acid or by addition of a chase solution containing glutamine, excess unlabeled ATP and bicarbonate. The intercept is decreased to about 50% if the excess unlabeled ATP is added prior to the addition of the glutamine. These are the expected results if the intercept reflects the reversible formation of enzyme-bound ADP and carboxyphosphate. Approximately 0.6 mol carbamoyl [32P]phosphate/mol enzyme is formed in these experiments when the pulse step is terminated by addition to the chase solution. The ATP molecule that provides the phosphoryl group of carbamoyl phosphate, therefore, also binds to the enzyme in the absence of ammonia or glutamine and reacts in the chase to give carbamoyl phosphate before it can dissociate from the enzyme. At 1 mM ATP, the binding of both ATP molecules is essentially complete at 2.5 s, but the dissociation of the ATP that yields carbamoyl phosphate is extremely slow (t(1/2) of about 6 min at 22 degrees C; HCO3-, 40 mM), although it is faster in the absence of bicarbonate. The extreme sequestration from the aqueous environment of this ATP allows the enzyme-ATP complex to be separated from the surrounding ATP by centrifugal gel filtration. After two successive steps of gel filtration through Sephadex G-50 equilibrated with unlabeled ATP and bicarbonate, the majority of the radioactivity remaining in the solution is bound to the enzyme and is released as [gamma-32P]ATP if acid is added, or is converted to carbamoyl [32P]phosphate by addition to chase solution, without concomitant release of 32Pi. K+ is necessary in the pulse solution, but not in the chase solution, to demonstrate this binding. These findings and other confirmatory experiments demonstrate conclusively that, in the presence of K+, both ATP molecules bind to the enzyme in the absence of ammonia or glutamine. The bound ATP that yields Pi in the overall reaction is replaced relatively rapidly by exchange and by hydrolysis in the bicarbonate-dependent ATPase activity of the enzyme, whereas the bound ATP that provides the phosphoryl group of carbamoyl phosphate is replaced very slowly. The temporal pattern of carbamoyl [32P]phosphate formation from [gamma-32P]ATP, in pulse/chase experiments in which a small concentration of ammonia is added to the pulse solution, shows that, in the normal enzyme reaction, this last ATP molecule binds to the enzyme before ammonia. These findings exclude a recently proposed mechanism [Kothe, M., Eroglu, B., Mazza, H., Samudera, H. & Powers-Lee, S. (1997) Proc. Natl Acad. Sci. USA 94, 12348-12353] in which a single molecule of ATP bound at the catalytic center phosphorylates bicarbonate and provides the phosphoryl group of carbamoyl phosphate. A mechanism in which a single ATP molecule binds, followed by the binding of bicarbonate and ammonia (from glutamine) and the release of Pi before the second molecule of ATP is bound is also excluded. We have previously reported very similar findings for carbamoyl-phosphate synthetase (ammonia), strongly suggesting that the different types of CPS share a common mechanism. The virtual sequestration of the ATP that provides the phosphoryl group of carbamoyl phosphate is consistent with a palmate-binding site, with the nucleotide bound within a beta-sheet sandwich, and a loop closure mechanism triggered by the binding of bicarbonate or the formation of carboxyphosphate.

Treatment of ornithine carbamoyltransferase from dolphin Stenella with pyridoxal phosphate, followed by reduction with NaBH4 resulted in complete loss of enzyme activity. The phosphate alone or the substrate analogue 2-aminovaleric acid moderately decreased the extent of inactivation, while carbamoyl phosphate plus 2-aminovaleric acid provided complete protection from inactivation. The partially inactivated enzyme showed K(m) values for substrates equivalent to those of native enzyme and lowered Kcat values. Two lysyl residues were substantially modified in the absence of ligands but only one of them was responsible for the inactivation of catalytic activity. Modification of a single subunit was sufficient to completely abolish the catalytic activity of the trimeric enzyme. The lysine involved has been identified as lysine 56 on the known primary structure of homologous human liver enzyme.

The reaction catalyzed by Escherichia coli aspartate transcarbamoylase (ATCase) proceeds through an ordered mechanism, in which carbamoylphosphate binds first, followed by aspartate; upon binding of this second substrate, the enzyme undergoes a concerted transition from a low-affinity T state to a high-affinity R state. In various studies, conflicting results were obtained concerning the existence of positive cooperativity for the first substrate, carbamoylphosphate. It is shown here that cooperativity for this substrate is only apparent. Indeed, saturation curves for carbamoylphosphate display sigmoidicity only if the aspartate concentration used is high enough to shift ATCase into the R state. Furthermore, it is shown that succinate, an unreactive aspartate analogue which is able to promote the T-->R conformational transition, also induces the appearance of cooperativity for carbamoylphosphate. Similar results were obtained in the course of continuous-flow-dialysis experiments, which show that the binding of carbamoylphosphate is apparently cooperative only in the presence of a concentration of succinate high enough to shift the enzyme into the R state. Taken together, these data show that the apparent cooperativity for carbamoylphosphate is not an intrinsic property of ATCase, as it only reflects the cooperativity for the second substrate, aspartate, as a consequence of the process of ordered substrate binding.

Ornithine carbamoyltransferases (OTCases) catalyse the formation of citrulline and phosphate from ornithine and carbamoylphosphate by a thermodynamically favoured reaction. In vivo, catabolic OTCase of Pseudomonas aeruginosa promotes the reverse reaction, the phosphorolysis of citrulline. Although the enzyme is assayed in vitro in the direction of citrulline synthesis, the enzyme cannot perform this reaction in vivo due to poor affinity for carbamoylphosphate and high cooperativity towards this substrate. Furthermore, the dodecameric catabolic OTCase is an allosteric enzyme; the enzyme is stimulated by nucleoside monophosphates and inhibited by polyamines (e.g. spermidine). A previous study showed that a modification of the C-terminus of the catabolic OTCase alters the homotropic cooperativity of the enzyme. We have now investigated the importance of the C-terminus for homotropic and heterotropic cooperativity by site-directed mutagenesis. Deletion of the C-terminal Ile335 residue strongly reduced cooperativity for carbamoylphosphate and sensitivity to spermidine. These properties were essentially restored when the two C-terminal amino acids (Asp334 and Ile335) were removed by deletion. However, in this variant enzyme, AMP failed to abolish carbamoylphosphate cooperativity completely, whereas the wild-type enzyme was rendered virtually non-cooperative by AMP. An extension of catabolic ornithine carbamoyltransferase by 15 amino acid residues interfered with both homotropic and heterotropic interactions and lowered the maximal velocity. All variant enzymes had the same dodecameric structure as the wild type and differed only slightly in affinity for the second substrate ornithine. A structural model of the dodecamer, at 0.3-nm resolution, suggests that the C-terminus could be involved in trimer/trimer interaction. We propose that modifications at the C-terminus alter the trimer/trimer interface and, in addition, removes the salt bridge His5-Ile335 within a monomer. These changes profoundly and indirectly modify the allosteric transition and consequently the interactions of the dodecamer with carbamoylphosphate and effectors.

Administration of either ammonia or glycine to both rats and mice results in an increased synthesis in the liver and urinary excretion of orotic acid. The two most relevant observations obtained are that carbamoyl phosphate synthesized inside the mitochondria is involved in the increased synthesis of orotic acid and that this latter process is almost completely abolished by cycloheximide and actinomycin D, inhibitors of protein and RNA synthesis. Orotic acid synthesis could be controlled by an induction-suppression mechanism. Inhibition of synthesis of excess orotic acid brought about by N-(phosphonacetyl)-L-aspartic acid but not by acivicin, suggests that glutamine-dependent cytosolic synthesis of carbamoyl phosphate, is not involved. Administration of ornithine together with glycine completely suppressed the synthesis of orotic acid, but promoted a twofold increase of urea excretion. The concentration of ornithine rather than that of carbamoyl phosphate or the activity of the enzymes involved, may represent a limiting factor controlling both the flux of ammonia in the urea cycle and the availability of mitochondrial carbamoyl phosphate for orotic acid synthesis. Two enzymes have been found to be induced by glycine: ornithine decarboxylase and aspartate transcarbamoylase (aspartate carbamoyltransferase). Both enzymes may contribute to the increase in orotic acid synthesis, aspartate transcarbamoylase more directly and ornithine decarboxylase by lowering the ornithine concentration. Ornithine decarboxylase activity was completely suppressed but that of aspartate transcarbamoylase was further increased by cycloheximide treatment. Inhibition of orotic acid biosynthesis by cycloheximide appears to be the result of a decreased availability in the cytosol of carbamoyl phosphate synthesized inside the mitochondria.

Carbamoyl-phosphate synthetase was purified from the deep-sea hyperthermophilic archaebacterium Pyrococcus abyssi. This enzyme appears to be monomeric and uses ammonium salts as nitrogen donor. Its activity is inhibited by some nucleotides that compete with ATP. In contrast with the carbamoyl-phosphate synthetases investigated so far, this enzyme is very resistant to high temperature. Its low molecular mass (46.6 kDa) and its catalytic properties suggest that the gene coding for this enzyme is a previously postulated ancestor, whose duplication gave the genes coding for carbamoyl-phosphate synthetases and carbamate kinases.

The capacity for L-arginine metabolism was studied in villus enterocytes isolated from pigs at birth, after 2-8 days suckling and after weaning. Immediately after birth, enterocytes were able to convert 1 mM L-citrulline, 2 mM L-glutamine or 1 mM L-ornithine to L-arginine. In 2-8-day-old animals, the net production of L-arginine from L-citrulline (2.00 +/- 0.45 nmol x 10(6) cells-1 x 30 min-1), or from L-ornithine (0.29 +/- 0.06 nmol x 10(6) cells-1 x 30 min-1) was similar to the values obtained at birth. Furthermore, 40% of L-arginine synthetized de novo from L-citrulline were released into the incubation medium. In 2-8-day-old animals, the production of L-arginine from L-glutamine represented only 5% of the production at birth (the latter being 0.73 +/- 0.15 nmol x 10(6) cells-1 x 30 min-1). In enterocytes isolated from post-weaned pigs, no significant production of L-arginine from either L-glutamine or L-ornithine was detected. In contrast, although the L-arginine production from L-citrulline was very low in post-weaned animals, it was significantly enhanced in the presence of L-glutamine, representing 23% of the production measured in suckling animals. The capacity of enterocytes to cleave L-arginine to L-ornithine and urea was very limited at birth, but was increased more than threefold in 2-day-old animals. This was concomitant with a marked increase in arginase activity. In post-weaned animals, the flux through arginase in intact enterocytes, and the arginase activity were both threefold higher than in 2-8-day-old animals. It is concluded that enterocytes isolated from neonatal pigs exhibit the capacity for a net production of L-arginine since the metabolism of this amino acid is oriented to anabolism rather than catabolism. The results are discussed in relation to L-arginine metabolism in the neonatal liver.

Pyridoxal phosphate, a competitive inhibitor of aspartate transcarbamylase, binds to six sites in the catalytic and to twelve sites in the regulatory subunits of this hexameric protein. The properties of its association to the active sites of the enzyme are very similar to those observed with one of its substrates, carbamyl phosphate. It tightly binds to one half of the sites in the absence of succinate, an analogue of the second substrate. Since pyridoxal phosphate can be linked covalently to the protein by reduction, the distribution of the high affinity binding sites on catalytic trimers was studied after dissociation of modified holoenzyme. Electrophoresis of isolated subunits under non-denaturing conditions revealed four distinct bands, corresponding to trimers containing 0 to 3 pyridoxal phosphate derivatives. The distribution among the four species as a function of ligand concentration in the absence of succinate indicates that in the native oligomer, pyridoxal phosphate (and by extrapolation, carbamyl phosphate) binds to both catalytic trimers, rather than to three sites on a single subunit.

The effect of exogenous adenine or uracil upon the de novo pathway for synthesis of pyrimidine nucleotides in Escherichia coli K12 was investigated. Parameters studied were levels of the enzymes carbamoyl phosphate synthase (EC 2.7.2.9), aspartate carbamoyltransferase (EC 2.1.3.2) and orotate phosphoribosyltransferase (EC 2.4.2.10) and the intermediates carbamoyl phosphate, aspartate and orotate, together with the contributions of exogenous uracil and aspartate to intracellular pyrimidine nucleotide. Taken with earlier data [Bagnara, A.S. & Finch, L. R. (1974) Eur. J. Biochem- 41, 421--430] on contents of UTP, CTP and 5-phosphoribosyl 1-diphosphate in cultures of this strain after the addition of adenine or uracil, the results obtained provide new insights into the regulatory mechanisms operating on the pathway in vivo. These insights enable evaluation of the contributions of such factors as limitation for a substrate, feed-back allosteric control by end products and enzyme repression/depression mechanisms. The evidence presented indicates that depressed levels of orotate phosphoribosyltransferase in E. coli K12 result in the wasteful ultilization of asparatate for excess synthesis of pyrimidine nucleotide precursors during balanced growth of the strain in minimal medium. Exogenous adenine increases the excessive accumulation of these precursors by lowering the intracellular content of 5-phosphoribosyl 1-diphosphate (Bagnara and Finch, 1974). This causes a decrease in the conversion of orotate to orotidine 5'-monophosphate, thus lowering the utilization or orotate and its precursors for synthesis of pyrimidine nucleotides. Further, since the contents of these nucleotide end products are thereby decreased (Bagnara nad Finch, 1974), theri feed-back on the early steps in the pathway is diminished and the production of the precursors is increased. It is postulated that growth of E. coli K12 under these conditions is limited by a compound that is metabolically related to precursors to aspartate.

Ornithine transcarbamoylase (OTC) is inactivated by liver lysosomes. Carbamoyl phosphate prevents the inactivation of OTC by lysosomes, while ATP, ADP, GTP, GDP 1,N6-ethenoadenosine 5'-triphosphate and particularly epsilon-ATP stimulate it. Both stimulation and protection occur at concentrations within the physiological range of ATP and carbamoyl phosphate. Inactivation of OTC is followed by extensive proteolysis. Since the inactivation is prevented by leupeptin, antipain and L-(tosylamido-2-phenyl)ethylchloromethyl ketone, the proteolytic susceptibility of OTC to lysosomes could be due to thiol endopeptidase(s). 1,N6-Ethenoadenosine 5'-triphosphate also markedly increases OTC susceptibility to trypsin and elastase. ATP analogs had no stimulatory effect on OTC inactivation by lysosomes; none of the inhibitors of ATPases tested inhibited the ATP effect. The ATP stimulation does not require Mg2+. These findings indicate a new role for ATP, GTP and related nucleotides in protein breakdown. The ATP, ADP, GTP, GDP stimulation, together with the carbamoyl protection of OTC, agree well with the molecular plasticity hypothesis model.

Blockade of a metabolic pathway by interaction of a drug with a particular ‘target enzyme’ results in depletion of essential end-products of the pathway and accumulation of intermediates prior to the blockade. Metabolic resistance to a particular drug can arise if the substrate of the inhibited enzyme accumulates to levels sufficiently high to compete effectively with the inhibitor, leading to restoration of full activity of the metabolic pathway after a transitory delay. Such resistance has recently been demonstrated in vitro for the interaction of the tight-binding inhibitor N-phosphonacetyl-l-aspartate (PAcAsp) with the aspartate transcarbamoylase activity of the trifunctional protein which initiates pyrimidine biosynthesis in mammals [Christopherson, R. I. and Jones, M. E. (1980) J. Biol. Chem. 255, 11381–11395]. Carbamoyl phosphate, the product of the carbamoyl phosphate synthetase activity of this trifunctional protein, accumulates to a sufficiently high concentration that the inhibitory effect of PAcAsp is effectively abolished. We have developed a theoretical model for metabolic resistance which quantitatively accounts for these experimental data. This model has been used to simulate the interaction between the following potential or proven anti-cancer drugs and their target enzyme, under conditions similar to those which would occur in vivo: PAcAsp with aspartate transcarbamoylase; various OMP analogues [the 5′-monophosphates of 6-azauridine, pyrazofurin and 1-(β-d-ribofuranosyl)-barbituric acid] with OMP decarboxylase; 5-fluorodeoxyUMP with thymidylate synthase; methotrexate with dihydrofolate reductase; and deoxycoformycin with adenosine deaminase.

1. The interrelationship between the two carbamoyl phosphate pools in intact hepatocytes and intact liver was studied with respect to de novo pyrimidine synthesis by use of selective inhibitors of the mitochonodiral and the cytosolic carbamoyl-phosphate synthetase. 2. Inhibition of mitochondrial carbamoyl phosphate synthesis by 4-pentenoate was without effect on galactosamine-stimulated pyrimidine synthesis. Conditions favouring mitochondrial carbamoyl phosphate accumulation, like excess ammonium ions or L-norvaline, led to an increase in pyrimidine synthesis bypassing the feedback inhibition of cytosolic carbamoyl-phosphate synthetase by UTP. 3. A stimulation of pyrimidine synthesis was not observed when the carbamoyl phosphate accumulation was due to aspartate deficiency in the presence of aminooxyacetate. The full response of pyrimidine synthesis to excess ammonium ions was restored, even in the presence of aminooxyacetate, when aspartate was substituted. This is explained by an inhibition of aspartate carbamoyltransferase flux [in view of the Km (aspartate = 0.7 mmol/1) of this enzyme] resulting from a 90% decrease in aspartate tissue levels. 4. Acivicin, the inhibitor of cytosolic carbamoyl-phosphate synthetase, completely abolished the galactosamine-induced stimulation of pyrimidine synthesis, but was without effect on the stimulation of pyrimidine synthesis by ammonium ions and L-norvaline. 5. It is concluded that experimental changes in mitochondrial carbamoyl phosphate content exert effects on de novo pyrimidine synthesis; however, it is considered unlikely that relevant amounts of mitochondrial carbamoyl phosphate are used for pyrimidine synthesis under physiological conditions. In addition the data point to a potential regulatory role of aspartate in hepatic pyrimidine synthesis.

A single subcutaneous administration of sodium [14C] bicarbonate was given to anemic mice previously treated with acetylphenylhydrazine. 14C rapidly appeared in the arterial blood, reached a peak value in less than 3 min, and disappeared with a half-life of about 7 min. The tracer thus administered offered a quasi pulse-labeling technique. The radioactive intermediates and end products in the hyperplastic and hematopoietic spleen were separated by repeated chromatography on anion-exchange resin. A relatively large amount of radioactivity appeared in carbamoylaspartate (peak value at 5 min), while definite but low radioactivity was found in dihydroorotate and orotate plus its derivatives (∑ orotate). The 14C in the uracil 5′-nucleotide pool (∑UMP) reached a maximum at 30 min and then declined with a half-life of about 60 min. l-[ureido-14C] Citrulline or l-[guanidino-14C] arginine induced no significant labeling of uracil nucleotides. Steady-state levels of carbamoyl phosphate, carbamoylaspartate, ∑ orotate, and ∑UMP were 0.12, 69.0, 5.0, and 513 nmol/g spleen. The above observations were consistent with the notion that production of carbamoyl phosphate limits the pathway, under the conditions used. When mice Were pretreated with 6-azauridine, a potent inhibitor of orotate conversion to UMP, there was a marked enhancement in the tissue contents of the intermediates, as well as a considerable accumulation of the 14C label, thus demonstrating that intermediates do not readily permeate through cell membranes. Kinetic analysis of the 14C label in carbamoylaspartate, together with reasonable assumptions, gave a value of 0.43 μmol × h−1× g−1 for the flow rate of the pathway. This value was close to the apparent turnover rate of ∑UMP (0.36 μmol × h−1× g−1), calculated from the half-life of the decline of the radioactivity and the pool size of ∑UMP (0.52 μmol/g).

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.

The amount of control of the various steps involved in the citrulline synthesizing pathway in isolated rat-liver mitochondria incubated with saturating concentrations of ammonia and ornithine has been measured. 1 The flux control coefficient of carbamoyl-phosphate synthase (ammonia) was close to one. Control exerted by other steps in the pathway, including ornithine transcarbamoylase, carbonic anhydrase, ATP production and transport of ornithine, was negligible. 2 The high flux control coefficient of carbamoyl-phosphate synthetase is due to the low elasticity coefficient of this enzyme towards its product, intramitochondrial carbamoyl phosphate, in combination with a high elasticity coefficient of ornithine transcarbamoylase towards carbamoyl phosphate.

1 When NH3 was added to isolated rat-liver mitochondria incubated with succinate and bicarbonate, oxidation of succinate was stimulated to a greater extent than could be accounted for by the net formation of carbamoyl phosphate. 2 Measurement of the rate of incorporation of [14C]bicarbonate into carbamoyl phosphate, after the mito- chondria had been preincubated with NH3 and unlabelled bicarbonate, revealed that flux through carbamoyl-phosphate synthetase (ammonia) was much greater than the net formation of carbamoyl phosphate indicated. 3 It is concluded that part of the carbamoyl phosphate produced in the absence of ornithine is degraded. About 20% of the degradation can be accounted for by non-enzymatic reactions of carbamoyl phosphate outside the mitochondria. It is proposed that the remainder of the degradation of carbamoyl phosphate occurs by partial reversal of the reaction of carbamoyl-phosphate synthetase.

A procedure for the permeabilization of Escherichia coli cells was adapted to the in situ determination of the catalytic and regulatory properties of the enzymes responsible for the biosynthesis of carbamoyl phosphate and its utilization in the pyrimidine and arginine pathways. Differences in enzyme sensitivity to effectors and changes in pH dependence were observed. Partition of carbamoyl phosphate in the two metabolic pathways could be measured under conditions of substrate saturation. The results obtained will allow to test experimentally the theoretical predictions made by A. Goldbeter (1973) PhD thesis, Universite Libre de Bruxelles, on the distribution of carbamoyl phosphate and the oscillation of its intracellular concentration.

Aspartate transcarbamylase is purified from mung bean seedlings by a series of steps involving manganous sulphate treatment, ammonium sulphate fractionation, DEAE-cellulose chromatography, followed by a second ammonium sulphate fractionation and finally gel filtration on Sephadex-G 100. The enzyme is homogeneous on ultracentrifugation and on polyacrylamide gel electrophoresis. It functions optimally at 55°C. It has two pH optima, one at 8.0 and the other at 10.2. The enzyme follows Michaelis-Menten kinetics with l-aspartate as the variable substrate. However, it exhibits sigmoid saturation curves at both the pH optima when the concentration of carbamyl phosphate is varied. The enzyme is allosterically inhibited by UMP at both the pH optima. Increasing phosphorylation of the uridine nucleotide decreases the inhibitory effect. The enzyme is desensitized to inhibition by UMP on treatment with p-hydroxymercuribenzoate, gel electrophoresis indicating that the enzyme is dissociated by this treatment; the dissociated enzyme can be reassociated by treatment with 2-mercaptoethanol. The properties of the mung bean enzyme are compared with the enzyme from other sources.

1 Slices of spleen from anaemic mice were incubated with [14C]bicarbonate in the presence and absence of 6-azauridine and the amounts of 14C that entered the de novo pyrimidine biosynthetic pathway were assessed and compared. Compounds analyzed included carbamoylaspartate, dihydroorotate, orotate plus its derivatives, acid-soluble uracil and cytosine 5′-nucleotides, nucleic acid pyrimidines, free pyrimidine bases and nucleosides. As the intracellular levels of carbamoyl phosphate and acid-soluble deoxyribonucleotides are known to be relatively low, the radioactivities of these compounds were not measured. Degradation of labelled uridine was limited in this tissues, therefore the radioactivity of degradative products of pyrimidines was not considered. 2 When the slices were incubated with 0.5 mM 6-azauridine for 10 min and then with [14C]bicarbonate for an additional 10 min and 30 min, the sum of radioactivity found in the above compounds, which represents the total amount of 14C that entered the pyrimidine pathway, was 2.1 and 2.3 times greater than when the tissue slices were incubated in the absence of the analogue. 3 When the 14C distribution among the carbon atoms of the molecules of labelled carbamoylaspartate and uracil was investigated, we found that more than 90% of the total 14C in these compounds derived directly from carbamoyl phosphate and the remaining portion was from aspartate, either in the presence or absence of 6-azauridine. 4 There was no indication that 6-azauridine altered [14C]bicarbonate permeation through the cell membrane or its intracellular metabolism. 5 These results, along with the pattern of early intermediate accumulation seen in the presence of 6-azauridine, indicate that 6-azauridine stimulates the production of carbamoyl phosphate for the pyrimidine biosynthetic pathway in the mouse spleen. 6 Of the radioactive early intermediates which accumulated, only orotate, its derivatives (orotidine and orotidine 5′-monophosphate) or both appeared in the medium, presumably the result of leakage through the cell membranes. 7 Stimulation of the pyrimidine pathway was not observed in the case of Ehrlich ascites tumour cells incubated under similar conditions with 6-azauridine.

Carbamylation of hemoglobin in the intact erythrocyte proceeds more readily with cyanate than with carbamyl phosphate. Carbamylation of hemoglobins A and S with carbamyl phosphate is lower at pH 6 than at pH 7 and even more so in the presence of azide. Carbamylation of insulin and histone also proceeds more readily with cyanate than with carbamyl phosphate. Mg2+, which diminishes carbamyl phosphate decomposition to cyanate, decreases also histone carbamylation with carbamyl phosphate. At pH 3.9 and in the presence of azide no histone carbamylation with carbamyl phosphate could be detected. On the bases of these experiments it is concluded that carbamylation of hemoglobin and other proteins with carbamyl phosphate proceed through cyanate. Rapid incorporation of potassium [14C]cyanate and [14C]carbamyl phosphate into erythrocyte ghosts was obtained. A discussion of the above results in relation to the suggested use of hemoglobin in sickle cell disease is presented.