Carbon-13 nuclear magnetic resonance analysis of [1-13C]glucose metabolism in Trypanosoma cruzi. Evidence of the presence of two alanine pools and of two CO2 fixation reactions.
The non-invasive technique of 13C-nuclear magnetic resonance was applied to study glucose metabolism in vivo in Trypanosoma cruzi, the causative agent of American trypanosomiasis (Chagas' disease). It was found that under anaerobic conditions [1-13C]glucose undergoes a glycolytic pathway whose main metabolic products were identified as [3-13C]alanine, [2-13C]succinate and phosphoryl[1-13C]choline; [2-13C]alanine was also a minor metabolite. The addition of 70% 2H2O to the incubation mixture led to the formation of [3-13C, 3-2H]alanine derived from the prior incorporation of 2H+ into pyruvate. The existence of a [3-13C, 3-2H]pyruvate precursor, although not isolated, could be inferred from the formation of [2-13C, 2-2H]succinate in the same experiment. The latter derives from the CO2 fixation reaction on pyruvate or phosphoenolpyruvate to give malate, which is then converted to succinate through the fumarate intermediate step. The presence of [2-13C]alanine must be traced to a randomization of label at the malate-fumarate stage. Both [3-13C, 3-2H]alanine and [2-13C, 2-2H]succinate were excreted from the cells into the supernatant. When the cell pellet was lysed with perchloric acid it released [3-13C]alanine which was devoid of 2H+. Hence, T. cruzi has two alanine pools: one which incorporates 2H+ from the 2H2O present in the medium and excretes alanine into the latter, and another which is impervious to 2H+ exchange. The fixation of CO2 on a C3 precursor was confirmed by incubation of the T. cruzi cells with [1-13C]glucose and sodium [13C]bicarbonate which led to the formation of [1,2-13C2]succinate (Jcc = 51.8 Hz). Incubation with sodium [13C]bicarbonate and [13C]glucose led to the formation of [1-13C]succinate (182.5 ppm) derived from the 13CO2 fixation on the C3 precursor, and of phosphoryl[1-13C]choline (59.39 ppm) which revealed the presence in T. cruzi of a reductive pathway of CO2 which is independent of the CO2 fixation reaction. The formation of phosphoryl[1-13C]choline from [1-13C]glucose should be attributed to 13CO2 liberated from the former by glucose-6-phosphate dehydrogenase.
Frydman
B
B
Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Argentina.
de los Santos
C
C
Cannata
J J
JJ
Cazzulo
J J
JJ
D000409:Alanine / Q000378:metabolism*; D000818:Animals; D002245:Carbon Dioxide / Q000378:metabolism*; D002247:Carbon Isotopes; D005947:Glucose / Q000378:metabolism*; D006019:Glycolysis; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008954:Models, Biological; D014349:Trypanosoma cruzi / Q000378:metabolism*
D002247:Carbon Isotopes; D002245:Carbon Dioxide; D005947:Glucose; D000409:Alanine