Physiological And Metabolic Magnetic Resonance Studies

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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.