INTERMEDIARY METABOLISM OF LEISHMANIA

利什曼原虫的中间代谢

• 批准号: 3140280
• 负责人: Jacob J Blum
• 金额: $ 18.94万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-08-01 至 1994-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3140280
• 关键词: Leishmania; adenosine triphosphate; aerobiosis; anaerobiosis; carbon dioxide; communicable disease control; density gradient ultracentrifugation; electron microscopy; electron probe spectrometry; enzyme mechanism; freeze etching; fructose phosphate; glucose 6 phosphate; glucose metabolism; intracellular parasitism; ion exchange chromatography; leishmaniasis; microorganism metabolism; molecular site; nuclear magnetic resonance spectroscopy; oxygen tension; phosphates; phosphoenolpyruvate; phosphotransferases

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.

核磁共振（NMR）光谱研究表明， 原生动物寄生虫硕大利什曼原虫的前鞭毛体与[1- 13 C]葡萄糖作为唯一碳源，存在空气或氧气释放 乙酸盐、丙酮酸盐、琥珀酸盐和少量甘油进入 缓冲盐溶液。在完全厌氧条件下，葡萄糖 消费几乎停止（“反向”巴斯德效应），除非二氧化碳是 目前，在这种情况下，琥珀酸是主要产品，甘油生产 增加，丙氨酸和D-乳酸也产生。这是第一 已知能产生乳酸盐D-异构体的寄生虫。比较研究 显示所有四种测试的利什曼原虫都只产生D-乳酸。 路易氏锥虫的血流形式使D-和L-乳酸，和T。 冈比亚布鲁氏菌仅产生L-异构体。这些观察引起了许多 关于D-乳酸盐形成的重要性的问题 锥虫，以及参与控制锥虫的调节机制， 在需氧与厌氧条件下形成的每种产物的量。我们 旨在测量某些关键中间代谢物的水平， 如ATP、葡萄糖-6-磷酸、磷酸烯醇丙酮酸和果糖-2，6 二磷酸盐，作为氧浓度的函数。知识 形成的各种产物水平的变化应该提供洞察力， 一些控制中间代谢的调节机制 在这些细胞中。31 P-NMR显示在样品中存在长链多磷酸盐。 前鞭毛体，我们已经证实了这一点，通过直接酶法测定， 多磷酸葡糖激酶电子探针X射线显微分析显示， 存在富含磷和镁的致密液泡。当 细胞在不存在碳源和不存在 磷酸盐，细胞内正磷酸盐和焦磷酸盐水平 增加这一增长是否部分是由于利用了 聚磷酸盐仍有待确定。研究将使用 酶测定、电子探针X射线微量分析和31 p-NMR 光谱学，以获得关于 多磷酸盐代谢以及在过程中发生的代谢变化 好氧到厌氧的转变。