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方法为完整系统、灌注器官、细胞悬液中的代谢和生理过程的研究以及细胞提取物的检查提供了独特的方法。细胞阳离子在激素信号传导中发挥重要作用，并且还参与细胞损伤的介导。细胞溶质阳离子和其他感兴趣的参数的细胞内指示剂的发展对细胞生物学领域产生了重大影响。我们最近开发了几种基于氟喹诺酮结构的Mg 2+指示剂，并一直在评估这些指示剂在各种细胞系统中的使用。我们最近将我们的氟化喹诺酮指示剂加载到红细胞中，并能够通过监测19 F NMR信号来跟踪镁浓度的变化。还使用荧光喹诺酮衍生物在叙利亚胚胎细胞中测定了细胞溶质Mg 2+水平。除了对细胞离子的研究外，最近的研究还集中在硼酸盐及其对γ-谷氨酰转肽酶（γ-GT）的影响，更普遍的是，对丝氨酸蛋白酶的影响。由硼酸盐和丝氨酸的组合的γ-谷氨酰转肽酶的抑制被证明是由于组装的过渡态类似物在酶的活性位点。这种抑制剂的结构类似物，L-2-氨基-4-硼基丁酸（L-ABBA）的合成和发现是一个有效的γ-GT的抑制剂。γ-GT的生理作用的阐明经常依赖于使用阿西维辛，这种酶的不可逆抑制剂。然而，阿西霉素也抑制许多其他酶，特别是参与嘌呤和嘧啶核苷酸生物合成的酶。已发现Acivicin调节多种细胞反应，包括生长、骨髓成熟和凋亡。为了评估γ-GT对这些过程的贡献，我们使用L-ABBA作为更具选择性的γ-GT抑制剂。这些研究表明，这些影响的acivicin不涉及γ-GT。最近，我们已经证明，形成一个三元复合物，涉及丝氨酸蛋白酶，硼酸盐，和各种醇是一个更普遍的现象胰蛋白酶，并可用于设计抑制剂，这一类的其他酶。