RESUMO
13C-nuclear magnetic resonance (NMR) spectroscopy was used to investigate the products of glycerol and acetate metabolism released by Leishmania braziliensis panamensis promastigotes and also to examine the interaction of each of these substrates with glucose or alanine. The NMR data were supplemented by measurements of the rates of oxygen consumption and substrate utilization, and of 14CO2 production from 14C-labeled substrate. Cells incubated with [2-13C]glycerol released acetate, succinate and D-lactate in addition to CO2. Cells incubated with acetate released only CO2. More succinate C-2/C-3 than C-1/C-4 was released from both [2-13C]glycerol and [2-13C]glucose, indicating that succinate was formed predominantly by CO2 fixation followed by reverse flux through part of the Krebs cycle. Some redistribution of the position of labeling was also seen in alanine and pyruvate, suggesting cycling through pyruvate/oxaloacetate/phosphoenolpyruvate. Cells incubated with combinations of 2 substrates consumed oxygen at the same rate as cells incubated with 1 or no substrate, even though the total substrate utilization had increased. When promastigotes were incubated with both glycerol and glucose, the rate of glucose consumption was unchanged but glycerol consumption decreased about 50%, and the rate of 14CO2 production from [1,(3)-14C]glycerol decreased about 60%. Alanine did not affect the rates of consumption of glucose or glycerol, but decreased 14CO2 production from these substrates by increasing flow of label into alanine. Although glucose decreased alanine consumption by 70%, it increased the rate of 14CO2 production from [U-14C]- and [l-14C]alanine by about 20%.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Acetatos/metabolismo , Alanina/metabolismo , Glucose/metabolismo , Glicerol/metabolismo , Leishmania braziliensis/metabolismo , Leishmania/metabolismo , Animais , Espectroscopia de Ressonância Magnética , Oxirredução , Consumo de OxigênioRESUMO
Leishmania braziliensis promastigotes incubated anaerobically produce D-lactate from glucose, ribose, and methylglyoxal, but not from glycerol, alanine, or pyruvate, suggesting the presence of glyoxalases I and II but the absence of D-lactate dehydrogenase. Further support for this is shown by: (1) conversion of methylglyoxal to D-lactate in sonicates of promastigotes in the presence of reduced glutathione, (2) utilization of phenylglyoxal at rates comparable to methylglyoxal, (3) lack of utilization of exogenously supplied D-lactate by promastigotes under aerobic conditions. Sonicates of promastigotes catalyze the conversion of dihydroxyacetone phosphate to methylglyoxal, suggesting the presence of methylglyoxal synthase. Whereas the rate of production of D-lactate from glucose is much greater under anaerobic conditions, the rate from methylglyoxal is independent of oxygen tension, indicating that control of flux through the methylglyoxal pathway occurs at, or before, methylglyoxal synthase.
Assuntos
Carbono-Oxigênio Liases , Lactatos/biossíntese , Lactoilglutationa Liase/metabolismo , Leishmania braziliensis/metabolismo , Leishmania/metabolismo , Liases/metabolismo , Tioléster Hidrolases/metabolismo , Alanina/metabolismo , Anaerobiose , Animais , Glucose/metabolismo , Glicerol/metabolismo , Leishmania braziliensis/efeitos dos fármacos , Leishmania braziliensis/enzimologia , Movimento/efeitos dos fármacos , Fenilglioxal/farmacologia , Aldeído Pirúvico/metabolismo , Aldeído Pirúvico/farmacologia , Piruvatos/metabolismo , Ácido Pirúvico , Ribose/metabolismo , SonicaçãoRESUMO
Leishmania braziliensis panamensis promastigotes were incubated with glucose as the sole carbon source. About one-fifth of the glucose consumed under aerobic conditions was oxidized to CO2. Nuclear magnetic resonance studies with [1-13C]glucose showed that the other products released were succinate, acetate, alanine, pyruvate, and lactate. Under anaerobic conditions, lactate output increased, glycerol became a major product, and, surprisingly, glucose consumption decreased. Enzymatic assays showed that the lactate formed was D(-)-lactate. The release of alanine during incubation with glucose as the sole carbon source suggested that appreciable proteolysis occurred, consistent with our observation that a large amount of ammonia was released under these conditions. The discoveries that D-lactate is a product of L. braziliensis glucose catabolism, that glycerol is produced under anaerobic conditions, and that the cells exhibit a "reverse" Pasteur effect open the way for detailed studies of the pathways of glucose metabolism and their regulation in this organism.
Assuntos
Glucose/metabolismo , Glicerol/biossíntese , Glicólise , Lactatos/biossíntese , Leishmania braziliensis/metabolismo , Leishmania/metabolismo , Aerobiose , Anaerobiose , Animais , Ácido Láctico , Espectroscopia de Ressonância MagnéticaRESUMO
Raising the temperature of a log-phase culture of Leishmania braziliensis panamensis promastigotes from 26 degrees C to 34 degrees C resulted in formation of a culture containing 85% ellipsoidally shaped forms after 1.5 h. The temperature-induced ellipsoidal forms decreased in size but persisted in high proportion (85-95%) for at least 12 h at 34 degrees C. Recovery from the ellipsoidal forms to a culture containing 85-95% promastigotes was observed after returning the temperature to 26 degrees C. The time required for recovery increased markedly with the duration of the preceding heat treatment, up to about 70 h for a 12-h heat treatment.