Physiological And Metabolic Magnetic Resonance Studies

核磁共振方法为研究完整系统、灌流器官、细胞悬浮液中的代谢和生理过程以及通过检查细胞提取物提供了一种独特的方法。细胞阳离子在激素信号转导中起重要作用，也参与细胞损伤的调节。细胞内阳离子指示剂和其他感兴趣的参数的发展对细胞生物学领域产生了重大影响。我们最近开发了几个基于氟喹诺酮结构的镁指示剂，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，并通过监测19F核磁共振信号来跟踪镁浓度的变化。使用一种荧光喹诺酮衍生物也测定了叙利亚仓鼠胚胎细胞中的胞浆镁离子水平。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伽马-谷氨酰转肽酶(伽马-GT)的影响，更广泛地说，对丝氨酸蛋白酶的影响。硼酸盐和丝氨酸的结合对谷氨酰转肽酶的抑制作用是通过在酶的活性部位组装过渡态类似物来实现的。合成了该缓蚀剂的结构类似物L-2-氨基-4-硼丁酸(L-ABBA)，发现其对γ-GT有很强的抑制作用。对γ-GT的生理作用的阐明常常依赖于该酶的一种不可逆转的抑制剂Acivicin的使用。然而，Acivicin也抑制许多其他酶，特别是那些与嘌呤和嘧啶核苷酸生物合成有关的酶。Acivicin被发现可以调节多种细胞反应，包括生长、髓系成熟和凋亡。为了评价γ-GT在这些过程中的贡献，我们使用了L-ABBA作为一种更具选择性的伽马-GT抑制剂。这些研究表明，阿昔维星的这些作用不涉及伽马-GT。最近，我们证明了丝氨酸蛋白酶、硼酸盐和各种醇形成的三元复合体是胰酶的一种更普遍的现象，可以用来设计这类其他酶的抑制剂。

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.