Chemical relaxation in a chemiosmotic-coupled system: driving the calcium adenosinetriphosphatase with...

Phase-lifetime spectroscopy has been used to measure chemical relaxation processes in a chemiosmotic-coupled system. In this experiment the calcium ATPase and bacteriorhodopsin were coreconstituted into phospholipid vesicles. Upon illumination the bacteriorhodopsin pumps protons into the vesicles and forms a membrane potential. This membrane potential alters the activity of the internal calcium and thus perturbs the equilibrium of ATP hydrolysis/synthesis coupled to calcium transport. Mechanically chopping the actinic light provides a periodic perturbation to the system, and small response signals can be observed by using phase-sensitive detection. It is shown that this periodic perturbation occurs about a steady-state membrane potential that is independent of chopping frequency. The amplitude dispersion curve for the fluorescence of a calcium indicator was observed and analyzed in terms of the relaxation time for the ATPase-catalyzed calcium transport. Thus, this technique provides a method of measuring ion transport kinetics against a constant chemiosmotic potential. The calcium ATPase showed a single relaxation time on this time scale. The dependence of this relaxation time on ADP and phosphate concentration was measured and analyzed with a random sequential mechanism. This analysis gave dissociation constants for ADP and phosphate of 3.2 mM and 1.4 mM, respectively. These binding steps are followed by slow isomerization steps with forward and reverse rate constants (in the direction of ATP synthesis) of 67 s-1 and 227 s-1, respectively. These results demonstrate that highly accurate kinetic data can be obtained with this modulation relaxation technique.