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 previously developed several Mg2+ indicators based on the fluoroquinolone structures, and have been evaluating their use in various cell systems. One of our fluorinated quinolone indicators was loaded into red blood cells, and were able to follow changes in magnesium concentration by monitoring the 19F NMR signals. However, the NMR characteristics of these indicators are not ideal, since in most cases, the fluorine resonances are subject to intermediate exchange at physiological temperatures. We recently have been able to synthesize a fluoropyridine Mg2+ indicator which exhibits considerably better NMR properties. 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. During the past year, we have obtained both NMR and crystallographic evidence for the formation of ternary and quaternary complexes involving alcohols, borate, and serine proteases. Such structures provide a basis for understanding the physiological effects of boron, as well as for the development of new enzyme inhibitors, analogous to our previous success with an inhibitor of gamma-glutamyl transpeptidase. Finally, in collaboration with the Cardiac physiology group (LST), we have recently used the fluorinated indicator TFBAPTA to demonstrate gender differences in the handling of calcium by the sarcoplasmic reticulum.
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