Darling T N, Davis D G, London R E, Blum J J
Department of Cell Biology, Duke University Medical Center, Durham, NC 27710.
Mol Biochem Parasitol. 1989 Mar 1;33(2):191-202. doi: 10.1016/0166-6851(89)90033-9.
The products released by Leishmania major promastigotes incubated with [1-13C]glucose as sole exogenous carbon source were identified using nuclear magnetic resonance (NMR). Under aerobic (95% O2/5% CO2) conditions, acetate, succinate, and small amounts of pyruvate, D-lactate, and glycerol were released in addition to CO2. Under anaerobic (95% N2/5% CO2) conditions, the relative amounts of products formed changed and alanine was also released. The changes in the rates of glucose consumption and product formation during the aerobic to anaerobic transition were measured. Under hypoxic conditions (O2 less than 0.2%), glucose consumption was decreased by about 50%. Under completely anaerobic conditions (100% N2), glucose consumption almost ceased (a total reverse Pasteur effect). The inclusion of 5% CO2 in the gas phase restored hypoxic and anaerobic glucose consumption to the aerobic rate, and increased production of succinate, pyruvate, and D-lactate. Thus, CO2 and very low concentrations of O2 have strong regulatory effects on L. major glucose metabolism. A quantitative carbon balance showed that the NMR-identified products accounted for only about 25% of the glucose carbons consumed under aerobic conditions. CO2, measured as the release of 14CO2 from [U-14C]glucose, accounted for an additional 25% of the glucose consumed. About 11% of the glucose carbon was incorporated into trichloroacetic acid-insoluble products, mostly lipid. Large amounts of label from [U-14C]glucose were incorporated into the intracellular pools of alanine, glutamate, glutamine, and aspartate, indicating that CO2 from unlabeled amino acids contributed to the carbon balance. Under anaerobic conditions, all the glucose carbons consumed could be accounted for solely by the NMR-identified products.
以[1-13C]葡萄糖作为唯一外源碳源培养的硕大利什曼原虫前鞭毛体释放的产物，通过核磁共振（NMR）进行鉴定。在需氧（95% O2/5% CO2）条件下，除了CO2外，还释放出乙酸盐、琥珀酸盐以及少量的丙酮酸、D-乳酸和甘油。在厌氧（95% N2/5% CO2）条件下，形成的产物相对量发生变化，并且还释放出丙氨酸。测定了需氧到厌氧转变过程中葡萄糖消耗速率和产物形成速率的变化。在低氧条件下（O2小于0.2%），葡萄糖消耗减少约50%。在完全厌氧条件下（100% N2），葡萄糖消耗几乎停止（完全的巴斯德效应逆转）。气相中加入5% CO2可使低氧和厌氧条件下的葡萄糖消耗恢复到需氧速率，并增加琥珀酸盐、丙酮酸和D-乳酸的产生。因此，CO2和极低浓度的O2对硕大利什曼原虫的葡萄糖代谢具有强烈的调节作用。定量碳平衡表明，NMR鉴定的产物仅占需氧条件下消耗的葡萄糖碳的约25%。以[U-14C]葡萄糖释放的14CO2来衡量，CO2占消耗的葡萄糖的另外25%。约11%的葡萄糖碳被掺入三氯乙酸不溶性产物中，主要是脂质。大量来自[U-14C]葡萄糖的标记物被掺入丙氨酸、谷氨酸、谷氨酰胺和天冬氨酸的细胞内池中，表明未标记氨基酸产生的CO2对碳平衡有贡献。在厌氧条件下，消耗的所有葡萄糖碳仅由NMR鉴定的产物来解释。
