Compartmentation of intracellular nucleotides in mammalian cells

The important role of nucleotides in cellular metabolism requires that serious consideration be given to the question of the homogeneity or inhomogeneity of nucleotide pools in cells. The purpose of this review is to summarize the existing evidence for compartmentation of nucleotide pools, discuss the limitations of this evidence, and to discuss the implications of compartmentation for the interpretation of nucleotide concentration measurements. Evidence for nucleotide compartmentation comes from the following types of evidence: compartmentation of RNA precursors; compartmentation of deoxynucleoside triphosphates; mitochondrial compartmentation; the existence of tightly bound nucleotides; pools derived from alternative synthetic routes; compartmentation in cyclic nucleotide metabolism; channeling in the synthesis of pyrimidine nucleotides; and others. The types of evidence adduced for compartmentation will be considered critically and in detail, and alternative explanations considered, as well. Implications of the data and hypotheses on nucleotide compartmentation for the interpretation of nucleotide pool measurements in various types of experiments will be discussed.     

Studies of intracellular thymidine nucleotides. Relationship between the synthesis of deoxyribonucleic acid and the thymidine triphosphate pool in Escherichia coli K12.

The two types of mutant strains which show resistance to T-even phage infection have been isolated and been shown to have either a higher or lower ratio of dTDP-sugar to dTTP than that of the parent strains. The one with a higher ratio of dTDP-sugar to dTTP than the parents has a large dTDP-sugar pool and small dTTP pool, and a high level of dTDPG pyrophosphorylase activity. The other one, with a lower ratio of dTDP-sugar to dTTP than the parents, has a small dTDP-sugar pool and large dTTP pool, and a low or deficient level of this enzyme activity. They form an entirely mucoid colony in the synthetic agar plate. Mutant cells (Ter-6 and Ter-21) which have deficient dTDPG pyrophosphorylase activity show 2 -- 3 times higher activity of UDPG pyrophosphoyrlase than that of parent cells. The dTDPG pyrophosphorylase-deficient mutants (Ter-15 and Ter-21) have a 3 -- 4 times higher concentration of dTTP and a faster rate of DNA synthesis and cell division than those of parent strains in growth with external thymine. The dTDPG pyrophosphorylase constitutive mutant (Ter-4) has a 0.5 -- 0.33 smaller dTTP pool and a slower rate of DNA synthesis and cell division than those of parent cells grown in the same medium. In the Ter-15 and Ter-21 mutants, the intracellular dTTP-dependent DNA synthesis rapidly disappeared in thymine suboptimal concentration, but the Ter-4 mutant maintained its dTTP-dependent DNA synthesis over a 20 muM concentration of external thymine. In high concentration (100 muM) of external thymidine, the thymidine effects on the intracellular dTTP concentration do not significantly appear in these enzyme-deficient mutants (Ter-15 and Ter-21). Also, the concentration of intracellular dTTP in the cell growth with external thymidine is 2.5 times greater than that with external thymine in these enzyme-deficient mutants (Ter-15 and Ter-21).     

Utilization of exogenous thymidine by Chlamydia psittaci growing in the thymidine kinase-containing and thymidine kinase-deficient L cells.

The incorporation of [3H]thymidine into the deoxyribonucleic acid (DNA) of Chlamydia psittaci (strain 6BC) growing in thymidine kinase (adenosine 5'-triphosphate-thymidine 5'-phosphotransferase, EC 1.7.1.21)-containing L cells, L(TK+), and thymidine kinase-deficient L cells, LM(TK-), was examined by autoradiography. Label was detected over C. psittaci inclusions in L(TK+) but not LM(TK-) cells. No evidence for a chlamydia-specific thymidine kinase activity in either L(TK+) or LM(TK-) cells was obtained. Entry of [3H]thymidine into the DNA of C. psittaci growing in L(TK+) cells was quantitated by measuring label in purified C. psittaci. It was 265 times less efficient than entry into infected host cell DNA. It is concluded that low levels of exogenous thymidine are incorporated into the DNA of C. psittaci and that this incorporation is dependent on a fully competent host thymidine kinase activity. Evidence also is presented that L cells possess at least two thymidine kinase activities, both of which are capable of supplying thymidylate precursors for nuclear DNA synthesis.     

Actinidia deliciosa in vitro II. Growth and exogenous carbohydrates utilization by explants

Changes in sugar composition (sucrose, glucose and fructose) of medium, callus, stem and leaves of in vitro proliferating explants of Actinidia deliciosa C.F. Liang, Hayward were analyzed together with explant growth at 0, 15, 30, 45 and 60 days of culturing. Autoclaving hydrolyzes a small part of the initial sucrose of the medium into glucose and fructose. In presence of Actinidia explants the initial sucrose decreased to 32% after 15 days of culturing, to 4% after 30 days and to 0.08% at the end of the culture period (60 days). Sucrose increase in the explants did not parallel with its decrease in the medium. Sucrose presence in the explants was evident only during the last month of culturing. After 15 days of culturing a large increase of glucose and fructose was found in the medium but it did not equal the hydrolyzed sucrose. The level of these two monosaccharides remained stable in the medium until the 30th day, then significantly decreased in the second month of culture; neither were completely exhausted at the end of the culture. In the whole explant the highest amount of glucose and fructose was reached after 30 days of culturing.The balance of the three sugars in the medium-explant system, as % distribution of carbon atoms, showed a utilization throughout the whole culture period.Qualitative analyses performed on medium, callus and leaves at 0, 15, and 30 days of culturing revealed the presence of glucose and fructose only and no significant amounts of other hexoses or pentoses. Starch accumulation in the leaves was also observed throughout the culturing.Paper No. 724     

Schistosoma mansoni: utilization of exogenous metabolites by eggs in vitro

Schistosoma mansoni eggs were isolated from the livers of mice infected for 8 wk and were purified by a series of settling and sieving procedures. Aliquots of eggs were suspended in saline with added Eagle's minimal essential medium supplemented with NaHCO₃, glutamine, penicillin, and streptomycin to which a variety of radioisotope labelled metabolites were added. The uptake and utilization of tritiated thymidine demonstrated a high rate of DNA synthesis, particularly in the more immature eggs studied. RNA synthesis as shown with tritiated uridine was also significant. Large amounts of ¹⁴C-labelled isoleucine and arginine were incorporated into protein. Little glycolytic activity was demonstrated on prolonged incubation with ¹⁴C-labelled glucose. A high rate of catabolism of amino acids to CO₂ was observed, as was a very high rate of acetate metabolism. Degradation of radiolabelled glutamate after incubation of eggs with ¹⁴C acetate revealed labelling consistent with metabolism via the Krebs cycle. Thus, Schistosoma mansoni eggs utilize a wide variety of exogenous metabolites. They show active DNA and RNA metabolism, incorporation of amino acids into protein, and intermediary metabolism characterized by a low rate of glycolysis and an active Krebs cycle.     

Synthesis of thymidine nucleotides in Bacillus subtilis.

To investigate the synthesis of thymidine nucleotides in Bacillus subtilis, mutants that carried various combinations of thyA, thyB, and other mutations affecting pyrimidine metabolism were isolated. It was found that exogenously supplied deoxycytidine was converted to thymidine nucleotides. The present data suggest that deoxycytidine nucleotides are first deaminated to yield deoxyuridine nucleotides which can serve as substrates for both thyA- and thyB-coded synthetases. A deaminase activity for dCDP was found in crude extracts of B. subtilis. A mutant lacking the deaminase activity was unable to convert deoxycytidine nucleotides to thymidine nucleotides.     

Incorporation of thymidine into the DNA of actinomycetes. I. Incorporation of exogenous thymidine into the DNA of Thermoactinomyces vulgaris]

The incorporation of exogenous thymidine and thymine into acid-insoluble material of Thermoactinomyces vulgaris has been studied during germination and subsequent growth. Thymine is not incorporated. The incorporation of thymidine stops after a short time due to the rapid breakdown of thymidine to thymine and deoxyribose-1-phosphate by the inducible thymidine phosphorylase. Deoxyadenosine enhances the incorporation of thymidine as well as of thymine and prolongs the tine of uptake. Uridine stimulates only the incorporation of thymidine but not of thymine. These effects can be explained by the function of these substances within the salvage pathway. Deoxyadenosine acts as donor of deoxyribosyl groups being necessary for the conversion of thymine to thymidine by thymidine phosphorylase and uridine inhibits thymidine phosphorylase, and thereby it prevents the degradation of thymidine to thymine. Thymidine is incorporated into alkali-, RNase-and protease-stable, hot TCA-soluble and DNase-sensitive material. That means that the cellular DNA of T. vulgaris can be specifically labelled by radioactive thymidine in the presence of deoxyadenosine and uridine, respectively.     

Inhibition of enzyme induction in Pseudomonas aeruginosa by exogenous nucleotides.

Alkylsufatase induction in resting cell suspensions of P. aeruginosa was inhibited by exogenously supplied adenosine or by ATP (2mM). Adenine phosphate had no effect while AMP or ADP caused a slight stimulation of induction. The inhibitory effect of ATP required the presence of added Mg2+, was not reversed by cyclic-AMP (2mM), and was independent of the nature of the inducer. Of a number of other nucleoside triphosphates tested, only UTP (2mM) acted as an inhibitor of induction. These nucleotides at external concentrations of 6mM also inhibited alkysulfatase induction in actively growing cells.     

Effect of exogenous nucleotides on the candicidin fermentation.

Addition of cyclic-AMP (c-AMP) to Streptomyces griseus fermentations inhibited candicidin formation. In a phosphate-free resting cell system, c-AMP inhibited net candicidin formation and incorporation of labeled propionate and p-aminobenzoic acid into the antibiotic but did not inhibit protein synthesis. All nucleotides tested, regardless of the position of the phosphate ester, were effective inhibitors; nucleosides and free bases were not. Inhibition occurred whether the nucleotide was added early or late. The results indicate that inhibition of antibiotic formation by exogenous nucleotides, including cyclic nucleotides, is similar to the effect produced by inorganic phosphate.     

Protection of mammalian cells from diphtheria toxin by exogenous nucleotides.

Exogenous nucleotides were found to protect mammalian cells from the lethal effects of diphtheria toxin. Protective potency of a given nucleotide was base specific and phosphate chain length dependent. Full expression of protective potency required an intact nucleotide, but the effect did not appear to be mediated by nucleotide-induced phosphorylation. Nucleotides antagonized the binding of diphtheria toxin to its cell surface receptor in a manner that correlated with the degree of protection. It was concluded that cellular protection from diphtheria toxin by nucleotides results from inhibition of toxin-receptor binding and that nucleotides therefore may serve as valuable research tools for future studies.