Nuclear Magnetic Resonance (NMR) spectroscopy studies show that promastigotes of the protozoan parasite Leishmania major incubated with [1- 13C] glucose as sole carbon source in the presence of air or oxygen release acetate, pyruvate, succinate, and small amounts of glycerol into the buffered salt solution. Under fully anaerobic conditions, glucose consumption practically ceases (""""""""reverse"""""""" Pasteur effect) unless CO2 is present, in which case succinate is the major product, glycerol production increases, and alanine and D-lactate are also produced. This is the first parasite known to produce the D- isomer of lactate. Comparative studies show that all four species of Leishmania tested make only D-lactate ' Trypanosome lewisi bloodstream forms make both D- and L- lactate, and T. brucei gambiense makes only the L- isomer. These observations raise many questions concerning the significance of D-lactate formation in the trypanosomatids, and the regulatory mechanisms involved in controlling the amount of each product formed under aerobic versus anaerobic conditions. We intend to measure the levels of certain key intermediate metabolites such as ATP, glucose-6-phosphate, phosphoenolpyruvate, and fructose-2,6 bisphosphate, as a function of oxygen concentration. Knowledge of the changes in levels of the various products formed should provide insight into some of the regulatory mechanisms controlling intermediary metabolism in these cells. 3lP-NMR shows the presence of long chain polyphosphate in promastigotes, and we have confirmed this by a direct enzymatic assay using polyphosphate glucokinase. Electron probe X-ray microanalysis shows the presence of dense vacuoles rich in phosphorous and magnesium. When the cells are incubated in the absence of a carbon source and the absence of phosphate, the intracellular orthophosphate and pyrophosphate levels increase. Whether some of this increase resulted from utilization of the polyphosphate remains to be determined. Studies will be performed using enzymatic assays, electron probe X-ray microanalysis, and 31p-NMR spectroscopy to obtain information on the interrelation between polyphosphate metabolism and the metabolic changes that occur during the aerobic to anaerobic transition.
| Keegan, F P; Blum, J J (1993) Incorporation of label from acetate and laurate into the mannan of Leishmania donovani via the glyoxylate cycle. J Eukaryot Microbiol 40:730-2 |
| Blum, J J (1992) Effect of osmolality on 86Rb+ uptake and release by Leishmania donovani. J Cell Physiol 152:111-7 |
| Keegan, F P; Blum, J J (1992) Utilization of a carbohydrate reserve comprised primarily of mannose by Leishmania donovani. Mol Biochem Parasitol 53:193-200 |
| Keegan, F P; Blum, J J (1991) Changes in intracellular levels of fructose 2,6-bisphosphate and several glycolytic intermediates in Leishmania major promastigotes as a function of pO2. Mol Biochem Parasitol 47:161-6 |
