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Abstract Background E. coli cells are rich in thiamine, most of it in the form of the cofactor thiamine diphosphate (ThDP). Free ThDP is the precursor for two triphosphorylated derivatives, thiamine triphosphate (ThTP) and the newly discovered adenosine thiamine triphosphate (AThTP). While, ThTP accumulation requires oxidation of a carbon source, AThTP slowly accumulates in response to carbon starvation, reaching ~15% of total thiamine. Here, we address the question whether AThTP accumulation in E. coli is triggered by the absence of a carbon source in the medium, the resulting drop in energy charge or other forms of metabolic stress. Results In minimal M9 medium, E. coli cells produce AThTP not only when energy substrates are lacking but also when their metabolization is inhibited. Thus AThTP accumulates in the presence of glucose, when glycolysis is blocked by iodoacetate, or in the presence lactate, when respiration is blocked by cyanide or anoxia. In both cases, ATP synthesis is impaired, but AThTP accumulation does not appear to be a direct consequence of reduced ATP levels. Indeed, in the CV2 E. coli strain (containing a thermolabile adenylate kinase), the ATP content is very low at 37°C, even in the presence of metabolizable substrates (glucose or lactate) and under these conditions, the cells produce ThTP but not AThTP. Furthermore, we show that ThTP inhibits AThTP accumulation. Therefore, we conclude that a low energy charge is not sufficient to trigger AThTP accumulation and the latter can only accumulate under conditions where no ThTP is synthesized. We further show that AThTP production can also be induced by the uncoupler CCCP but, unexpectedly, this requires the presence of pyruvate or a substrate yielding pyruvate (such a D-glucose or L-lactate). Under the conditions described, AThTP production is not different when RelA or SpoT mutants are used. Conclusions In E. coli, AThTP accumulates in response to two different conditions of metabolic stress: lack of energy substrates (or inhibition of their metabolization) and uncoupled pyruvate oxidation. Both conditions prevent bacterial growth. There is no obvious link with the stringent response or catabolite repression.

Abstract Background Thiamine triphosphate (ThTP) exists in most organisms and might play a role in cellular stress responses. In E. coli, ThTP is accumulated in response to amino acid starvation but the mechanism of its synthesis is still a matter of controversy. It has been suggested that ThTP is synthesized by an ATP-dependent specific thiamine diphosphate kinase. However, it is also known that vertebrate adenylate kinase 1 catalyzes ThTP synthesis at a very low rate and it has been postulated that this enzyme is responsible for ThTP synthesis in vivo. Results Here we show that bacterial, as vertebrate adenylate kinases are able to catalyze ThTP synthesis, but at a rate more than 106-fold lower than ATP synthesis. This activity is too low to explain the high rate of ThTP accumulation observed in E. coli during amino acid starvation. Moreover, bacteria from the heat-sensitive CV2 strain accumulate high amounts of ThTP (>50% of total thiamine) at 37°C despite complete inactivation of adenylate kinase and a subsequent drop in cellular ATP. Conclusion These results clearly demonstrate that adenylate kinase is not responsible for ThTP synthesis in vivo. Furthermore, they show that E. coli accumulate large amounts of ThTP under severe energy stress when ATP levels are very low, an observation not in favor of an ATP-dependent mechanisms for ThTP synthesis.

In immature neurones, the steady-state intracellular Cl[sup -] concentration [Cl[sup -] ][sub i] is generally higher than expected for passive distribution, and this is believed to be due to Na[sup +] –K[sup +] -2Cl[sup –] co-transport. Here, we show that N2a neuroblastoma cells, incubated in HEPES-buffered NaCl medium maintain a [Cl[sup -] ][sub i] around 60 mm, two- to threefold higher than expected for passive distribution at a membrane potential of - 49 mV. When the cells were transferred to a Cl[sup -] -free medium, [Cl[sup -] ][sub i] decreased quickly (t[sub 1/2] < 5 min), suggesting a high Cl[sup -] permeability. When the intracellular ATP concentration was reduced to less than 1 mm by metabolic inhibitors, the initial rate of [sup 36] Cl[sup -] uptake was strongly inhibited (60–65%) while steady-state [Cl[sup -] ][sub i] decreased to 24 mm, close to the value predicted from the Nernst equilibrium. Moreover, after reduction of [ATP][sub i] and [Cl[sup -] ][sub i] by rotenone, the subsequent addition of glucose led to a reaccumulation of Cl[sup -] , in parallel with ATP recovery. Internal bicarbonate did not affect Cl[sup -] pumping, suggesting that Cl[sup -] /HCO[sub 3][sup –] exchange does not significantly contribute to active transport. Likewise, Na[sup +] –K[sup +] -2Cl[sup –] co-transport also appeared to play a minor role: although mRNA for the NKCC1 form of the co-transporter was detected in N2a cells, neither the initial rate of [sup 36] Cl[sup -] uptake nor steady-state [Cl[sup -] ][sub i] were appreciably decreased by 10 µm bumetanide or replacement of external Na[sup +] by choline. These results suggest that a highly active ATP-dependent mechanism, distinct from Na[sup +] –K[sup +] -2Cl[sup –] co-transport, is responsible for most of the inward Cl[sup -] pumping in N2a cells. [ABSTRACT FROM AUTHOR]

Several methods for the chemical synthesis of γ-32P-labeled and unlabeled nucleoside 5′-triphosphates and thiamine triphosphate (ThTP) have been described. They often proved unsatisfactory because of low yield, requirement for anhydrous solvents, procedures involving several steps or insufficient specific radioactivity of the labeled triphosphate. In the method described here, all these drawbacks are avoided. The synthesis of [γ-32P]ThTP was carried out in one step, using 1,3-dicyclohexyl carbodiimide as condensing agent for thiamine diphosphate and phosphoric acid in a dimethyl sulfoxide/pyridine solvent mixture. Anhydrous solvents were not required and the yield reached 90%. After purification, [γ-32P]ThTP had a specific radioactivity of 11 Ci/mmol and was suitable for protein phosphorylation. The method can also be used for the synthesis of [γ-32P]ATP of the desired specific radioactivity. It can easily be applied to the synthesis of unlabeled ThTP or ribo- and deoxyribonucleoside 5′-triphosphates. In the latter case, inexpensive 5′-monophosphate precursors can be used as reactants in a 20-fold excess of phosphoric acid. Deoxyribonucleoside 5′-triphosphates were obtained in 6 h with a yield of at least 70%. After purification, the nucleotides were found to be suitable substrates for Taq polymerase during polymerase chain reaction cycling. Our method can easily be scaled up for industrial synthesis of a variety of labeled and unlabeled triphosphoric derivatives from their mono- or diphosphate precursors. [Copyright &y& Elsevier]