NMR measurements of Ca2+ and H+ transport mediated by A23187 and reconstituted plasma membrane Ca(2+)-ATPase.
NMR-based assays for measuring the fluxes of Ca2+, H+, and ATP in liposomal systems are presented. The 19F NMR Ca(2+)-chelating molecule 5,5-difluoro-1,2-bis(o-amino-phenoxy)ethane-N,N,N',N'-tetraacetic acid (5FBAPTA) was trapped 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 general applicability of 5FBAPTA as a 19F NMR probe of active Ca2+ transport. 31P NMR time-courses were used to monitor simultaneously the ATP hydrolysing activity of the reconstituted human erythrocyte Ca(2+)-ATPase and the concomitant acidification of the reaction medium in a suspension of small unilamellar vesicles. Using an estimate of the extraliposomal buffering capacity, the H+/ATP coupling stoichiometry, in the presence of A23187, was estimated from the NMR-derived data at steady state; 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 thermodynamics model of the pump (accompanying paper in this issue). Importantly, NMR, in contrast to optical detection methods, can potentially register all fluxes and (electro)chemical gradients involved in the Ca(2+)-ATPase-mediated H+/Ca2+ counterport, in a single experiment.
Waldeck
A R
AR
Department of Biochemistry, University of Sydney, N.S.W., Australia.
Xu
A S
AS
Roufogalis
B D
BD
Kuchel
P W
PW
D000255:Adenosine Triphosphate / Q000378:metabolism; D001693:Biological Transport, Active; D000001:Calcimycin / Q000494:pharmacology*; D002118:Calcium / Q000493:pharmacokinetics*; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D002462:Cell Membrane / Q000201:enzymology; D002614:Chelating Agents; D004533:Egtazic Acid / Q000031:analogs & derivatives; D005461:Fluorine; D006859:Hydrogen / Q000493:pharmacokinetics*; D006863:Hydrogen-Ion Concentration; D007476:Ionophores / Q000494:pharmacology*; D008081:Liposomes / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008432:Mathematical Computing; D010758:Phosphorus; D011510:Proteolipids / Q000378:metabolism
D002614:Chelating Agents; D007476:Ionophores; D008081:Liposomes; D011510:Proteolipids; D010758:Phosphorus; D005461:Fluorine; D000001:Calcimycin; D004533:Egtazic Acid; D006859:Hydrogen; C039877:5,5'-difluoro-1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases; D002118:Calcium