NMR MEASUREMENTS OF CA2-MEMBRANE CA2+-ATPASE( AND H+ TRANSPORT MEDIATED BY A23187 AND RECONSTITUTED PLASMA)

NMR-based assays for measuring the fluxes of Ca2+, H+, and ATP in lipo somal systems are presented. The F-19 NMR Ca2+-chelating molecule 2-bi s(o-amino-phenoxy)ethane-N,N,N',N'-tetraacetic acid (5FBAPTA) was trap ped inside large unilamellar vesicles and used to monitor passive and A23187-mediated Ca2+ transport into them. The data were analyzed using progress curves of the transport reaction. They demonstrated the gene ral applicability of 5FBAPTA as a F-19 NMR probe of active Ca2+ transp ort. P-31 NMR time-courses were used to monitor simultaneously the ATP hydrolysing activity of the reconstituted human erythrocyte Ca2+-ATPa se and the concomitant acidification of the reaction medium in a suspe nsion of small unilamellar vesicles. Using an estimate of the extralip osomal buffering capacity, the H+/ATP coupling stoichiometry, in the p resence of A23187, was estimated from the NMR-derived data at steady s tate; it amounted to 1.4+/-0.3. This result is discussed with respect to the issue of molecular 'slip' in the context of a non-equilibrium t hermodynamics model of the pump (accompanying paper in this issue). Im portantly, NMR, in contrast to optical detection methods, can potentia lly register all fluxes and (electro)chemical gradients involved in th e Ca2+-ATPase-mediated H+/Ca2+ counterport, in a single experiment.