Physiological And Metabolic Magnetic Resonance Studies

生理和代谢磁共振研究

• 批准号: 6672986
• 负责人: Robert E London
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6672986
• 关键词: biological models; calcium channel blockers; cations; cellular pathology; cytoplasm; heart cell; ischemia; laboratory rabbit; method development; nitric oxide; nuclear magnetic resonance spectroscopy; perfusion; reagent /indicator; sarcoplasmic reticulum

NMR methods provide a unique approach for the investigation of metabolic and physiological processes in intact systems, perfused organs, cell suspensions, as well as by examination of cell extracts. Cellular cations play fundamental roles in hormonal signaling, and are also involved in the mediation of cell injury. The development of intracellular indicators for cytosolic cations and other parameters of interest has had a major impact on the field of cell biology. We have recently developed several Mg2+ indicators based on the fluoroquinolone structures, and have been evaluating the use of these in various cell systems. We recently loaded our fluorinated quinolone indicator into red blood cells, and were able to follow changes in magnesium concentration by monitoring the 19F NMR signals. Cytosolic Mg2+ levels were also determined in Syrian Hamster Embryo cells using a fluorescent quinolone derivative. In addition to the work on cellular ions, recent studies have also focused on borate and its effects on gamma-glutamyl transpeptidase (gamma-GT) and, more generally, on serine proteases. The inhibition of gamma-glutamyl transpeptidase by the combination of borate and serine was shown to result from assembly of a transition state analog at the active site of the enzyme. A structural analog of this inhibitor, L-2-amino-4-boronobutanoic acid (L-ABBA) was synthesized and found to be a potent inhibitor of gamma-GT. Elucidation of the physiological role of gamma-GT has frequently relied on the use of acivicin, an irreversible inhibitor of this enzyme. However, acivicin also inhibits many other enzymes, particularly those involved in purine and pyrmidine nucleotide biosynthesis. Acivicin has been found to modulate various cellular responses including growth, myeloid maturation and apoptosis. In order to evaluate the contribution of gamma-GT on these processes, we used L-ABBA as a more selective gamma-GT inhibitor. These studies have shown that these effects of acivicin do not involve gamma-GT. More recently, we have demonstrated that the formation of a ternary complex involving the serine protease, borate, and various alcohols is a more general phenomenon trypsin, and may be used to design inhibitors for other enzymes of this class.

NMR方法为研究完整系统，灌注器官，细胞悬浮液以及通过检查细胞提取物的代谢和生理过程提供了独特的方法。细胞阳离子在激素信号传导中起着基本作用，也参与细胞损伤的介导。胞质阳离子和其他感兴趣的参数的细胞内指标的发展对细胞生物学领域产生了重大影响。我们最近基于氟喹诺酮结构开发了几个MG2+指标，并一直在评估它们在各种细胞系统中的使用。最近，我们将氟化的喹诺酮指示剂加载到红细胞中，并通过监测19F NMR信号来遵循镁浓度的变化。还使用荧光奎诺酮衍生物在叙利亚仓鼠胚胎细胞中确定胞质Mg2+水平。除了在细胞离子方面的工作外，最近的研究还集中在硼酸盐及其对γ-谷氨酰基肽酶（Gamma-GT）的影响，更普遍地是丝氨酸蛋白酶。通过硼酸盐和丝氨酸的组合，抑制γ-谷氨酰基转肽酶的抑制是由酶的活性位点的过渡态类似物组装而成的。合成了该抑制剂L-2-Amino-4-核丁烷酸（L-ABBA）的结构类似物，并被发现是γ-GT的有效抑制剂。阐明γ-GT的生理作用经常依赖于这种酶的不可逆抑制剂Acivicin的使用。然而，阿无环蛋白还抑制许多其他酶，尤其是参与嘌呤和吡啶核苷核苷酸生物合成的酶。已发现阿无环素可以调节各种细胞反应，包括生长，髓样成熟和凋亡。为了评估γ-GT在这些过程中的贡献，我们使用L-ABBA作为更具选择性的伽玛GT抑制剂。这些研究表明，阿无环蛋白的这些作用不涉及γ-GT。最近，我们证明了涉及丝氨酸蛋白酶，硼酸盐和各种醇的三元络合物的形成是一种更普遍的现象，可以用于设计该类别其他酶的抑制剂。