STUDIES OF CARDIAC METABOLISM BY 13C AND 31P NMR

这个项目的主要目标是利用最近的进展 在31P和13C核磁共振(核磁共振)领域制造 大鼠心脏灌流后能量和代谢的光谱学研究 在不同的底材供应和工作条件下。重点将是 习惯于使用富含13C的底物来研究对 柠檬酸循环及其与苹果酸-天冬氨酸循环的相互作用 增减柠檬酸循环净池大小的机理 中间体。使用两颗完整的心脏将获得13C核磁共振谱 和高氯酸提取物。后者也将用于酶制剂。 代谢物的测定，以帮助识别和定量共振 从13C谱中的单个13C标记的碳原子中获得。 从13C特定标记模式提供的信息 中间商，以及对其总池大小的了解 由代谢分析确定的，将被用来构建一个流量模型 柠檬酸循环和相关的转氨基步骤。模型 将被用来检验关于通量调节的各种假说 不同底物供应条件下的柠檬酸循环 心脏在稳态和过渡状态下的功输出 循环通量不均匀时的状态。以下是主要的具体目标 将会得到解决。1.速率控制步骤的识别 柠檬酸循环及抑制剂对酶调控强度的评估 滴定研究。2.对存在和代谢的评估 代谢物区隔和底物通道的意义。3. 苹果酸-天冬氨酸循环中不同通量速率对细菌生长的影响 柠檬酸合成酶α-酮戊二酸对通量的调节 脱氢酶和天冬氨酸氨基转移酶。4.调查 导致组织内容物净变化的机制 柠檬酸循环中间体。5.乙酰乙酸酯对乳酸菌生长发育的影响 通过琥珀酸硫激酶和丙酸在助熔剂上的存在和缺失 从α-酮基-戊二酸到苹果酸的柠檬酸循环的调节。 6.复氧后柠檬酸循环功能的研究 缺血后心脏。

The major goal of this project is to take advantage of the recent advances made in the field of 31p and 13C nuclear magnetic resonances (NMR) spectroscopy to study the energetics and metabolism of perfused rat hearts under different conditions of substrate supply and work. Emphasis will be given to the use of 13C-enriched substrates to study regulation of the citric acid cycle, its interactions with the malate-asparate cycle and mechanisms for increasing or decreasing net pool sizes of citric acid cycle intermediates. 13C NMR spectra will be obtained using both intact hearts and perchloric acid extracts. The latter will also be used for enzymatic assay of metabolites to aid identification and quantitation of resonances obtained from individual 13C-labeled carbon atoms from the 13C spectra. The information provided from 13C specific labeling patterns of intermediates, together with knowledge of their total pool sizes as determined by metabolic analyses, will be used to construct a flux model of the citric acid cycle and interrelated transamination steps. The model will be used to test various hypotheses concerning regulation of flux in the citric acid cycle under different conditions of substrate supply and work output of the heart during the steady state as well as transition states when cycle flux is nonuniform. The following major specific aims will be addressed. 1. Identification of rate-controlling steps in the citric acid cycle and assessment of enzyme control strengths by inhibitor titration studies. 2. Assessment of the existence and metabolic significance of metabolite compartmention and substrate channeling. 3. Effects of varying flux rates through the malate-asparate cycle on the regulation of flux through citrate syntase, Alpha-ketoglutarate dehydrogenase and asparate aminotransferase. 4. Investigation of the mechanisms responsible for causing net changes of the tissue contents of citric acid cycle intermediates. 5. Effects of acetoacetate in the absence and presence of propionate on flux through succinate thiokinase and regulation of the citric acid cycle from Alpha-keto-glutarate to malate. 6. Investigation of citric acid cycle function in reoxygenated post-ischemic hearts.