Diphosphate concentration does not correlate with the level of inorganic diphosphatase in Escherichia coli.

Partial inhibition of DNA synthesis stimulates the production of inorganic diphosphatase in Escherichia coli but the changes in diphosphate (PPi) level observed did not correlate with the enzyme activity. An accumulation of PPi was observed in the presence of inhibitors of RNA synthesis or nucleotide synthesis. In the former case the level of the enzyme did not change but in the latter case it increased. Thus the amount of inorganic diphosphatase alone does not determine the concentration of PPi in E. coli.

Accumulation of pyrophosphate correlates with the increased level of nucleoside triphosphates in Escherichia coli.

We measured the concentration of nucleoside triphosphates and inorganic pyrophosphate in Escherichia coli in conditions where nucleotide synthesis or nucleic acid synthesis was inhibited. The inhibitors that brought about an accumulation of some of the four ribonucleoside triphosphates also increased the pyrophosphate level. In a pyrimidine auxotrophic strain uracil starvation led to simultaneous accumulation of ATP and pyrophosphate, and they both rapidly returned to normal level when starvation was relieved. These results indicate the possible involvement of pyrophosphate in the reactions leading to the accumulation of nucleoside triphosphates.

Accumulation of pyrophosphate in Escherichia coli. Relationship to growth and nucleotide synthesis.

Pyrophosphate (PPi) content of Escherichia coli is increased manyfold when the growth of the cells is limited by inhibition of the synthesis of nucleotides. The accumulated PPi is immediately degraded when inhibition is released and growth allowed to resume. Other tested nutritional deficiencies (starvation of carbon source, sulfate or an amino acid) fail to induce PPi accumulation.

A method for concentration of nucleoside triphosphates by coprecipitation with calcium fluoride.

A method for concentration of nucleoside triphosphates (NTP) is described. NTPs are quantitatively coprecipitated from the solution with calcium fluoride. The precipitate is separated by filtration through a membrane filter and NTPs are dissolved from the filter by immersing it in 0.5 N H2SO4. With this method also nucleoside diphosphates can be efficiently concentrated, but the method does not work with nucleoside monophosphates or cyclic AMP.

Hydroxyurea inhibits also the syntheses of thymine nucleotides and methionine in the cells of Escherichia coli.

Hydroxyurea not only inhibited the reduction of ribonucleotides, but also gave rise to need of exogenous methionine and thymidine for full growth in the cultures of Escherichia coli K12. In vitro the drug inhibited the formation of 5,10-methylenetetrahydrofolate by serine hydroxymethyltransferase in the cell extracts, which suggests that this reaction is a secondary target of hydroxyurea in the cells of E. coli. The syntheses of thymine nucleotides and methionine were also the most sensitive targets of hydroxylamine in the cultures of E. coli.

Regulation of inorganic pyrophosphatase in Escherichia coli: relationship between the synthesis of inorganic pyrophosphatase and the thymidine triphosphate pool.

The activities of inorganic pyrophosphatase, thymidine kinase and thymidine phosphorylase were measured in Ter-mutants of E. coli K12 which have a higher or a lower dTTP pool than the parent strain. The levels of inorganic pyrophosphatase and thymidine kinase were changed in the same direction and that of thymidine phosphorylase in the opposite direction in these mutants.

The intracellular concentration of pyrophosphate in the batch culture of Escherichia coli.

We applied our colorimetric PPi determination method [Heinonen, J., Honkasalo, S. and Kukko, E. (1981) Anal. Biochem. 117, 293-300] to bacterial cultures and measured the intracellular concentration of PPi in the batch culture of Escherichia coli. The cells growing in minimal medium contained about 2.5 nmol PPi/mg protein (0.5 mM). There was no extracellular PPi. A similar concentration of PPi was found also in cells growing in minimal medium enriched by amino acid mixture.

Effect of penicillins on the level of inorganic pyrophosphatase in Escherichia coli K 12.

The relative rate of DNA synthesis increases over control level and the synthesis of inorganic pyrophosphatase is stimulated when cells of E. coli are cultured in the presence of penicillins which inhibit cell division and cause filamentation of the cells. In contrast to that in temperature sensitive cell division mutants filamentation at 42 degrees C does not change the DNA/cell mass ratio and the synthesis of inorganic pyrophosphatase is not stimulated. This suggests that the change in the synthesis of the enzyme is not correlated with the filamentation but is a consequence of the supernormal DNA/cell mass ratio that appears in the presence of penicillins.