Colon tumor: enzymology of the neoplastic program

The biochemical strategy of colon tumor was investigated by comparing the enzymic programs of glycolysis, pentose phosphate production and purine and pyrimidine biosynthesis and degradation in liver, normal colon mucosa and transplantable colon adenocarcinoma in the mouse. In normal colon mucosa the carbohydrate and pentose phosphate enzymes were 2- to 9-fold higher in specific activity than those in liver. Among the enzymes of CTP synthesis, CTP synthetase was the rate-limiting one in both liver and colon. In colon tumor CTP synthetase, OMP decarboxylase, uracil phosphoribosyltransferase and thymidine kinase activities increased to 927, 863, 597 and 514% of activities of normal colon. In contrast, the activity of the catabolic enzymes, dihydrothymine dehydrogenase and uridine phosphorylase, decreased to 51 and 25%. The ratios of activities of uridine kinase/uridine phosphorylase and thymidine kinase/dihydrothymine dehydrogenase were elevated 6- and 10-fold. The activity of the key purine synthetic enzyme, glutamine PRPP amidotransferase, increased 7-fold and the opposing rate-limiting enzyme of purine catabolism, xanthine oxidase, decreased to 7%. The ratio of amidotransferase/xanthine oxidase was elevated to 8, 150%. Activities of glucose-6-phosphate dehydrogenase and transaldolase did not increase, but that of pyruvate kinase was elevated to 154%. Similar enzymic programs were observed in a transplantable adenocarcinoma of the colon in the rat. The alterations in gene expression in colon tumor manifested in an integrated pattern of enzymic imbalance indicate the display of a program, a segment of which is shared with rat and human liver and kidney tumors. These alterations in gene expression should confer selective advantages to colon tumor cells. The striking increases in the activities of CTP synthetase, OMP decarboxylase, glutamine PRPP amidotransferase and thymidine kinase mark out these enzymes as potentially sensitive targets for combination chemotherapy by specific inhibitors of these enzyme activities.     

In situ regulation of mammalian CTP synthetase by allosteric inhibition

The regulatory role of the allosteric site of CTP synthetase on flux through the enzyme in situ and on pyrimidine nucleotide triphosphate (NTP) pool balance was investigated using a mutant mouse T lymphoblast (S49) cell line which contains a CTP synthetase refractory to complete inhibition by CTP. Measurements of [3H]uridine incorporation into cellular pyrimidine NTP pools as a function of time indicated that CTP synthesis in intact wild type cells was markedly inhibited in a cooperative fashion by small increases in CTP pools, whereas flux across the enzyme in mutant cells was much less affected by changes in CTP levels. The cooperativity of the allosteric inhibition of the enzyme was greater in situ than in vitro. Exogenous manipulation of levels of GTP, an activator of the enzyme, indicated that GTP had a moderate effect on enzyme activity in situ, and changes in pools of ATP, a substrate of the enzyme, had small effects on CTP synthetase activity. The consequences of incubation with actinomycin D, cycloheximide, dibutyryl cyclic AMP, and 6-azauridine on the flux across CTP synthetase and on NTP pools differed considerably between wild type and mutant cells. Under conditions of growth arrest, an intact binding site for CTP on CTP synthetase was required to maintain a balance between the CTP and UTP pools in wild type cells. Moreover, wild type cells failed to incorporate H14CO3- into pyrimidine pools following growth arrest. In contrast, mutant cells incorporated the radiolabel at a high rate indicating loss of a regulatory function. These results indicated that uridine nucleotides are important regulators of pyrimidine nucleotide synthesis in mouse S49 cells, and CTP regulates the balance between UTP and CTP pools.     

Glutamine-dependent carbamoyl-phosphate synthetase and control of pyrimidine biosynthesis in the parasitic helminth Schistosoma mansoni.

1. Carbomoyl-phosphate synthetase in Schistosoma mansoni utilizes L-glutamine as well as ammonia as nitrogen donor but does not require N-acetyl-L-glutamate for the activity. 2. The enzyme activity was inhibited by UDP, UTP, ADP and AMP, among which UDP was the most effective. 3. Aspartate carbamoyltransferase and dihydroorotase were also found and copurified with the synthetase. 4. Relative activities among these three enzymes were 1:30-60:3-8 throughout the purification. 5. These results suggest that the synthetase plays a key role in the control of pyrimidine biosynthesis de novo.     

3'-O-(4-benzoyl)benzoylcytidine 5'-triphosphate. A substrate and photoaffinity label for CMP-N-acetylneuraminic acid synthetase

A photoreactive, radiolabeled pyrimidine nucleotide, 3'-O-(4-benzoyl)benzoylcytidine 5'-triphosphate was synthesized from benzoylbenzoic acid and radiolabeled CTP. Benzoylbenzoyl-[5-3H]CTP could substitute for CTP, in an enzymatic reaction with N-acetylneuraminic acid catalyzed by Escherichia coli or rat liver CMP-NeuAc synthetase, to yield radiolabeled benzoyl-benzoyl-CMP-NeuAc. E. coli CMP-NeuAc synthetase could be specifically radiolabeled using benzoylbenzoyl-[alpha-32P]CTP as a photoaffinity label. This specific covalent binding occurred using enzyme preparations of different degrees of purity. These results suggest that benzoylbenzoic acid derivatives of pyrimidines should be of general use in the identification and active site mapping of pyrimidine-requiring proteins and enzymes. These include glycosyltransferases, sugar nucleotide synthetases, and transporters, and enzymes participating in the conjugation of bile acids and biosynthesis of nucleic acids and choline nucleotides.     

Growth-dependent regulation of mammalian pyrimidine biosynthesis by the protein kinase A and MAPK signaling cascades

The carbamoyl phosphate synthetase domain of the multifunctional protein CAD catalyzes the initial, rate-limiting step in mammalian de novo pyrimidine biosynthesis. In addition to allosteric regulation by the inhibitor UTP and the activator PRPP, the carbamoyl phosphate synthetase activity is controlled by mitogen-activated protein kinase (MAPK)- and protein kinase A (PKA)-mediated phosphorylation. MAPK phosphorylation, both in vivo and in vitro, increases sensitivity to PRPP and decreases sensitivity to the inhibitor UTP, whereas PKA phosphorylation reduces the response to both allosteric effectors. To elucidate the factors responsible for growth state-dependent regulation of pyrimidine biosynthesis, the activity of the de novo pyrimidine pathway, the MAPK and PKA activities, the phosphorylation state, and the allosteric regulation of CAD were measured as a function of growth state. As cells entered the exponential growth phase, there was an 8-fold increase in pyrimidine biosynthesis that was accompanied by a 40-fold increase in MAPK activity and a 4-fold increase in CAD threonine phosphorylation. PRPP activation increased to 21-fold, and UTP became a modest activator. These changes were reversed when the cultures approach confluence and growth ceases. Moreover, CAD phosphoserine, a measure of PKA phosphorylation, increased 2-fold in confluent cells. These results are consistent with the activation of CAD by MAPK during periods of rapid growth and its down-regulation in confluent cells associated with decreased MAPK phosphorylation and a concomitant increase in PKA phosphorylation. A scheme is proposed that could account for growth-dependent regulation of pyrimidine biosynthesis based on the sequential action of MAPK and PKA on the carbamoyl phosphate synthetase activity of CAD.     

Characterization of CMP-N-acetylneuraminic acid synthetase of group B streptococci.

The capsular polysaccharide is a critical virulence factor for group B streptococci associated with human infections, yet little is known about capsule biosynthesis. We detected CMP-Neu5Ac synthetase, the enzyme which activates N-acetylneuraminic acid (Neu5Ac, or sialic acid) for transfer to the nascent capsular polysaccharide, in multiple group B streptococcus serotypes, all of which elaborate capsules containing Neu5Ac. CMP-Neu5Ac synthetase isolated from a high-producing type Ib strain was purified 87-fold. The enzyme had apparent Km values of 7.6 for Neu5Ac and 1.4 for CTP and a pH optimum of 8.3 to 9.4, required magnesium, and was stimulated by dithiothreitol. This is the first characterization of an enzyme involved in group B streptococcus capsular polysaccharide biosynthesis.     

Cytotoxic mechanisms of glutamine antagonists in mouse L1210 leukemia

The glutamine antagonists, acivicin (NSC 163501), azaserine (NSC 742), and 6-diazo-5-oxo-L-norleucine (DON) (NSC 7365), are potent inhibitors of many glutamine-dependent amidotransferases in vitro. Experiments performed with mouse L1210 leukemia growing in culture show that each antagonist has different sites of inhibition in nucleotide biosynthesis. Acivicin is a potent inhibitor of CTP and GMP synthetases and partially inhibits N-formylglycineamidine ribotide (FGAM) synthetase of purine biosynthesis. DON inhibits FGAM synthetase, CTP synthetase, and glucosamine-6-phosphate isomerase. Azaserine inhibits FGAM synthetase and glucosamine-6-phosphate isomerase. Large accumulations of FGAR and its di- and triphosphate derivatives were observed for all three antagonists which could interfere with the biosynthesis of nucleic acids, providing another mechanism of cytotoxicity. Acivicin, azaserine, and DON are not potent inhibitors of carbamyl phosphate synthetase II (glutamine-hydrolyzing) and amidophosphoribosyltransferase in leukemia cells growing in culture although there are reports of such inhibitions in vitro. Blockade of de novo purine biosynthesis by these three antagonists results in a "complementary stimulation" of de novo pyrimidine biosynthesis.     

The effect of cyclic nucleotides on purine biosynthesis and the induction of PRPP synthetase during lymphocyte activation

The activation of lymphocytes has been used to study the regulation of mammalian gene expression. Concanavalin A (Con A) added to mouse spleen lymphocytes in serum-free medium leads to an increase in the rate of DNA synthesis as great as 1000 fold, commencing 20 hr after its addition. Prior to 20 hr, the rate of purine synthesis increases 10–100 fold as measured by accumulation of the purine intermediate, formyl glycineamide ribonucleotide (FGAR). Addition of dibutyryl cyclic GMP to the lymphocyte suspensions results in a 10 fold increase in the rate of DNA synthesis in the absence of Con A and enhances both purine synthesis and DNA synthesis in its presence. The activity of phosphoribosyl pyrophosphate synthetase (PRPP synthetase), an enzyme central to purine and pyrimidine biosynthesis, is increased 2–10 fold during the activation. The increase begins to appear 8 hr after Con A addition and requires concomitant protein synthesis. The induced PRPP synthetase activity is stimulated by the presence of cyclic GMP in the enzyme assay. Addition of dibutyryl cyclic AMP to Con A-stimulated lymphocytes inhibits FGAR production, the stimulation of DNA synthesis, and the appearance of cyclic GMP-sensitive PRPP synthetase. These studies suggest that cyclic nucleotides play a significant role in the molecular mechanism of lymphocyte activation, the regulation of purine biosynthesis, and of eucaryotic genetic expression.     

Pyrimidine nucleotides impair phosphoribosylpyrophosphate (PRPP) synthetase subunit aggregation by sequestering magnesium. A mechanism for the decreased PRPP synthetase activity in hereditary erythrocyte pyrimidine 5'-nucleotidase deficiency

The activity of phosphoribosylpyrophosphate (PRPP) synthetase (ATP: D-ribose-5-phosphate pyrophosphotransferase, EC 2.7.6.1) is decreased in the erythrocyte in hereditary pyrimidine 5'-nucleotidase (P5N) deficiency. Given the increased pyrimidine nucleotide content of the P5N-deficient erythrocyte, we evaluated the effects of prototypic pyrimidine nucleotides on the activity of PRPP synthetase. In normal hemolysate a 1.0 mM combination of cytidine tri-, di- and monophosphate (CTP/CDP/CMP) inhibited PRPP synthetase activity and changed the ribose 5-phosphate (R5P) saturation curve from a hyperbola to a biphasic shape. Untreated crude hemolysate from P5N-deficient erythrocytes showed a biphasic R5P kinetic curve. Since the activity of PRPP synthetase is dependent on its state of subunit aggregation, we examined PRPP synthetase subunit aggregation using gel permeation chromatography. P5N-deficient erythrocytes had a decreased absolute amount of aggregated PRPP synthetase and almost a total loss of disaggregated PRPP synthetase. Using normal hemolysate, 1 mM CTP/CDP/CMP interfered with the ability of 1.0 mM ATP and 2.0 mM MgCl2 to promote PRPP synthetase subunit aggregation. Increasing the MgCl2 to 6.0 mM overcame the inhibitory effect of CTP/CDP/CMP. Thus, the decreased PRPP synthetase activity of the P5N-deficient erythrocyte is due, at least in part, to the ability of the accumulated pyrimidine nucleotides to sequester magnesium and to interfere with the subunit aggregation of PRPP synthetase.     

Enxymology of human colon tumors

The biochemical strategy of human colon adenocarcinoma was studied by elucidating the enzymic programs of pyrimidine biosynthesis and degradation, glycolysis, pentose phosphate production, and galactose metabolism in normal colon mucosa and in 9 cases of primary colon adenocarcinoma. Enzymic activities were determined in the 100,000 X $\text{g}$ supernatant fluid with spectrophotometric or isotopic assays under optimum conditions yielding linear kinetics. In the human colon tumors the activities of enzymes of the $\text{de}$$\text{novo}$ pyrimidine biosynthesis, CTP synthetase, OMP decarboxylase, and orotate phosphoribosyltransferase, were increased to 348, 183, and 201% of those of normal human colon mucosa. The activities of the salvage pathway enzymes, thymidine kinase, uracil phosphoribosyltransferase and uridine kinase, were increased to 331, 254 and 281%. By contrast, the activity of the catabolic enzyme, uridine phosphorylase, was decreased to 69%. The ratio of activities of uridine kinase/ uridine phosphorylase was elevated to 564%. The activities of the key glycolytic enzymes, hexokinase and pyruvate kinase, and those of pentose phosphate production, glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and transaldolase, increased to 348, 209, 262, 156, and 180% respectively. The activity of the first committed enzyme of galactose utilization, galactokinase, was increased to 315%. The enzymic program of human primary colonic adenocarcinoma was similar in most respects to that which we observed in chemically-induced, transplantable adenocarcinomas of the colon in mouse and in rat (4). The reprogramming of gene expression in human colon tumor provides an increased capacity for biosynthesis of pyrimidines and ribose 5-phosphate, and for utilization of the glycolytic pathway and of galactose. These alterations in gene expression should confer selective advantages to the human colon tumor cells. The marked elevations in the activities of the salvage enzymes, uridine-cytidine kinase and thymidine kinase, explain in part the failure to obtain good therapeutic results with inhibitors of the $\text{de}$$\text{novo}$ pathway and account, in part at least, for the clinical difficulties encountered in the treatment of colon tumors. The elevated activities of CTP synthetase, OMP decarboxylase, uridine-cytidine kinase and thymidine kinase mark out these enzymes as targets for combination chemotherapy. Through such enzyme-pattern-targeted chemotherapy the drug treatment of human colon tumors should be improved.     

Expression of Human CTP synthetase in Saccharomyces cerevisiae reveals phosphorylation by protein kinase A

CTP synthetase (EC 6.3.4.2, UTP:ammonia ligase (ADP-forming)) is an essential enzyme in all organisms; it generates the CTP required for the synthesis of nucleic acids and membrane phospholipids. In this work we showed that the human CTP synthetase genes, CTPS1 and CTPS2, were functional in Saccharomyces cerevisiae and complemented the lethal phenotype of the ura7Delta ura8Delta mutant lacking CTP synthetase activity. The expression of the CTPS1- and CTPS2-encoded human CTP synthetase enzymes in the ura7Delta ura8Delta mutant was shown by immunoblot analysis of CTP synthetase proteins, the measurement of CTP synthetase activity, and the synthesis of CTP in vivo. Phosphoamino acid and phosphopeptide mapping analyses of human CTP synthetase 1 isolated from (32)P(i)-labeled cells revealed that the enzyme was phosphorylated on multiple serine residues in vivo. Activation of protein kinase A activity in yeast resulted in transient increases (2-fold) in the phosphorylation of human CTP synthetase 1 and the cellular level of CTP. Human CTP synthetase 1 was also phosphorylated by mammalian protein kinase A in vitro. Using human CTP synthetase 1 purified from Escherichia coli as a substrate, protein kinase A activity was dose- and time-dependent, and dependent on the concentrations of CTP synthetase 1 and ATP. These studies showed that S. cerevisiae was useful for the analysis of human CTP synthetase phosphorylation.     

Pyrimidine Nucleotide-Stimulated Thromboxane A2 Release from Cultured GLIA

1. Uridine triphosphate (UTP), uridine diphosphate (UDP), cytidine triphosphate (CTP), and deoxythymidine triphosphate (TTP) caused concentration-dependent increases in the release of thromboxane A₂ (TXA₂) from cultured glia prepared from the newborn rat cerebral cortex. Although each of the pyrimidine nucleotides displayed similar potencies, CTP and TTP were considerably less effective than either UTP or UDP. The purine nucleotide ATP was equally as potent as the pyrimidine nucleotides but was marginally less effective than either UTP or UDP.2. The ability of UTP, UDP, TTP, and CTP to promote TXA₂ release from cultured glia was inhibited in a concentration-dependent manner by suramin and was markedly reduced when incubations were performed either in Ca²⁺-free medium or on cultures which had been maintained in serum-free growth medium for 4 days prior to experimentation.3. Challenges with UTP and UDP in combination were found to elicit a response which was no different from the effects of these nucleotides alone; in addition, their effects were reversed by the phospholipase A₂ inhibitor ONO-RS-082. A slight reduction in UTP-and UDP-stimulated TXA₂ release was observed in cultures grown in the presence of leucine methyl ester, a treatment reported to limit microglial survival.4. These results suggest that glia are targets for extracellular pyrimidine nucleotides and that their ability to release eicosanoids from these cells may be important in the brain's response to damage.     

Biosynthesis of CMP-N,N'-diacetyllegionaminic acid from UDP-N,N'-diacetylbacillosamine in Legionella pneumophila

Legionaminic acid is a nine-carbon alpha-keto acid that is similar in structure to other members of the sialic acid family that includes neuraminic acid and pseudaminic acid. It is found as a component of the lipopolysaccharide in several bacterial species and is perhaps best known for its presence in the O-antigen of the causative agent of Legionnaires' disease, Legionella pneumophila. In this work, the enzymes responsible for the biosynthesis and activation of N, N'-diacetyllegionaminic acid are identified for the first time. A cluster of three L. pneumophila genes bearing homology to known sialic acid biosynthetic genes ( neuA,B,C) were cloned and overexpressed in Escherichia coli. The NeuC homologue was found to be a hydrolyzing UDP- N, N'-diacetylbacillosamine 2-epimerase that converts UDP- N, N'-diacetylbacillosamine into 2,4-diacetamido-2,4,6-trideoxymannose and UDP. Stereochemical and isotopic labeling studies showed that the enzyme utilizes a mechanism involving an initial anti elimination of UDP to form a glycal intermediate and a subsequent syn addition of water to generate product. This is similar to the hydrolyzing UDP- N-acetylglucosamine 2-epimerase (NeuC) of sialic acid biosynthesis, but the L. pneumophila enzyme would not accept UDP-GlcNAc as an alternate substrate. The NeuB homologue was found to be a N, N'-diacetyllegionaminic acid synthase that condenses 2,4-diacetamido-2,4,6-trideoxymannose with phosphoenolpyruvate (PEP), although the in vitro activity of the recombinant enzyme (isolated as a MalE fusion protein) was very low. The synthase activity was dependent on the presence of a divalent metal ion, and the reaction proceeded via a C-O bond cleavage process, similar to the reactions catalyzed by the sialic acid and pseudaminic acid synthases. Finally, the NeuA homologue was shown to possess the CMP- N, N'-diacetyllegionaminic acid synthetase activity that generates the activated form of legionaminic acid used in lipopolysaccharide biosynthesis. Together, the three enzymes constitute a pathway that converts a UDP-linked bacillosamine derivative into a CMP-linked legionaminic acid derivative.     

Cell cycle-dependent regulation of pyrimidine biosynthesis

De novo pyrimidine biosynthesis is activated in proliferating cells in response to an increased demand for nucleotides needed for DNA synthesis. The pyrimidine biosynthetic pathway in baby hamster kidney cells, synchronized by serum deprivation, was found to be up-regulated 1.9-fold during S phase and subsequently down-regulated as the cells progressed through the cycle. The nucleotide pools were depleted by serum starvation and were not replenished during the first round of cell division, suggesting that the rate of utilization of the newly synthesized nucleotides closely matched their rate of formation. The activation and subsequent down-regulation of the pathway can be attributed to altered allosteric regulation of the carbamoyl-phosphate synthetase activity of CAD (carbamoyl-phosphate synthetase-aspartate carbamoyltransferase-dihydroorotase), a multifunctional protein that initiates mammalian pyrimidine biosynthesis. As the culture approached S-phase there was an increased sensitivity to the allosteric activator, 5-phosphoribosyl-1-pyrophosphate, and a loss of UTP inhibition, changes that were reversed when cells emerged from S phase. The allosteric regulation of CAD is known to be modulated by MAP kinase (MAPK) and protein kinase A (PKA)-mediated phosphorylations as well as by autophosphorylation. CAD was found to be fully autophosphorylated in the synchronized cells, but the level remained invariant throughout the cycle. Although the MAPK activity increased early in G(1), the phosphorylation of the CAD MAPK site was delayed until just before the onset of S phase, probably due to antagonistic phosphorylation by PKA that persisted until late G(1). Once activated, pyrimidine biosynthesis remained elevated until rephosphorylation of CAD by PKA and dephosphorylation of the CAD MAPK site late in S phase. Thus, the cell cycle-dependent regulation of pyrimidine biosynthesis results from the sequential phosphorylation and dephosphorylation of CAD under the control of two important signaling cascades.     

Organisation and sequence determination of glutamine-dependent carbamoyl phosphate synthetase II in Toxoplasma gondii

Carbamoyl phosphate synthetase II encodes the first enzymic step of de novo pyrimidine biosynthesis. Carbamoyl phosphate synthetase II is essential for Toxoplasma gondii replication and virulence. In this study, we characterised the primary structure of a 28kb gene encoding Toxoplasma gondii carbamoyl phosphate synthetase II. The carbamoyl phosphate synthetase II gene was interrupted by 36 introns. The predicted protein encoded by the 37 carbamoyl phosphate synthetase II exons was a 1,687 amino acid polypeptide with an N-terminal glutamine amidotransferase domain fused with C-terminal carbamoyl phosphate synthetase domains. This bifunctional organisation of carbamoyl phosphate synthetase II is unique, so far, to protozoan parasites from the phylum Apicomplexa (Plasmodium, Babesia, Toxoplasma) or zoomastigina (Trypanosoma, Leishmania). Apicomplexan parasites possessed the largest carbamoyl phosphate synthetase II enzymes due to insertions in the glutamine amidotransferase and carbamoyl phosphate synthetase domains that were not present in the corresponding gene segments from bacteria, plants, fungi and mammals. The C-terminal allosteric regulatory domain, the carbamoyl phosphate synthetase linker domain and the oligomerisation domain were also distinct from the corresponding domains in other species. The novel C-terminal regulatory domain may explain the lack of activation of Toxoplasma gondii carbamoyl phosphate synthetase II by the allosteric effector 5-phosphoribosyl 1-pyrophosphate. Toxoplasma gondii growth in vitro was markedly inhibited by the glutamine antagonist acivicin, an inhibitor of glutamine amidotransferase activity typically associated with carbamoyl phosphate synthetase II, guanosine monophosphate synthetase, or CTP synthetase.     

Regulation of pyrimidine nucleotide biosynthesis in Pseudomonas synxantha

Regulation of pyrimidine nucleotide biosynthesis in Pseudomonas synxantha ATCC 9890 was investigated and the pyrimidine biosynthetic pathway enzyme activities were affected by pyrimidine supplementation in cells grown on glucose or succinate as a carbon source. In pyrimidine-grown ATCC 9890 cells, the activities of four de novo enzymes could be depressed which indicated possible repression of enzyme synthesis. To learn whether the pathway was repressible, pyrimidine limitation experiments were conducted using an orotate phosphoribosyltransferase (pyrE) mutant strain identified in this study. Compared to excess uracil growth conditions for the succinate-grown mutant strain cells, pyrimidine limitation of this strain caused dihydroorotase activity to increase about 3-fold while dihydroorotate dehydrogenase and orotidine 5'-monophosphate decarboxylase activities rose about 2-fold. Regulation of de novo pathway enzyme synthesis by pyrimidines appeared to be occurring. At the level of enzyme activity, aspartate transcarbamoylase activity in P. synxantha ATCC 9890 was strongly inhibited in vitro by pyrophosphate, UTP, ADP, ATP, CTP and GTP under saturating substrate concentrations.     

Control of fatty acid synthetase mRNA levels in rat liver by insulin, glucagon, and dibutyl cyclic AMP

The quantity of translatable fatty acid synthetase mRNA in liver of rats subjected to different hormonal states was determined with a rabbit reticulocyte lysate cell-free translation system. Both membrane-free polysomal and total cellular poly (A)-containing RNA were translated. The level of translatable fatty acid synthetase mRNA was 11-fold or more lower in livers of diabetic rats than in similar animals treated with insulin. In contrast, both glucagon and dibutyl cyclic AMP caused a 3-fold reduction over controls in the amount of translatable fatty acid synthetase mRNA in livers of animals refed a fat-free diet for 12 hr. These changes are consistent with the previously reported alterations in the relative rates of fatty acid synthetase synthesis measured in vivo. This suggests that the changes in the amount of fatty acid synthetase that occur in liver in response to the above hormonal changes are primarily due to changes in the amount of mRNA coding for this enzyme.     

De novo synthesis of pyrimidine nucleotides by Helicobacter pylori

The incorporation of pyrimidine nucleotide precursors into Helicobacter pylori and the activities of enzymes involved in their synthetic pathways were investigated by radioactive tracer analysis and ³¹P nuclear magnetic resonance spectroscopy. The bacterium was found to take up aspartate and bicarbonate and to incorporate carbon atoms from these precursors into its genomic DNA. Orotate, an intermediate of de novo pyrimidine biosynthesis, and uracil and uridine, precursors for pyrimidine pathways, were also incorporated by the micro-organism. Radiolabelled substrates were used to assess the activities of aspartate transcarbamoylase, orotate phosphoribosyltransferase, orotidylate decarboxylase, CTP synthetase, uracil phosphoribosyltransferase, thymidine kinase and deoxycytidine kinase in bacterial lysates. The study provided evidence for the presence in H. pylori of an operational de novo pathway, and a less active salvage pathway for the biosynthesis of pyrimidine nucleotides.     

Use of synthetic lethal mutants to clone and characterize a novel CTP synthetase gene in Saccharomyces cerevisiae

In the pyrimidine biosynthetic pathway, CTP synthetase catalyses the conversion of uridine 5′-triphosphate (UTP) to cytidine 5′-triphosphate (CTP). In the yeast Saccharomyces cerevisiae, the URA7 gene encoding this enzyme was previously shown to be nonessential for cell viability. The present paper describes the selection of synthetic lethal mutants in the CTP biosynthetic pathway that led us to clone a second gene, named URA8, which also encodes a CTP synthetase. Comparison of the predicted amino acid sequences of the products of URA7 and URA8 shows 78% identity. Deletion of the URA8 gene is viable in a haploid strain but simultaneous presence of null alleles both URA7 and URA8 is lethal. Based on the codon bias values for the two genes and the intracellular concentrations of CTP in strains deleted for one of the two genes, relative to the wild-type level, URA7 appears to be the major gene for CTP biosynthesis. Nevertheless, URA8 alone also allows yeast growth, at least under standard laboratory conditions.