Measurement of the Electrochemical Proton Gradient in

The pH gradient and membrane potential of submitochondrial particles from bovine heart were estimated by the uptake of ["C]ethylamine and ["'Cl]perchlorate, using filtration through a glass fiber prefilter and Millipore filter without washing to separate the vesicles from the medium. An external volume probe of ['HI sucrose was also used. Internal volume of the vesicles was measured by the extent of uptake of glucose, which equilibrates slowly across the membrane. The electrochemical potential gradient of H+ (A,&+) calculated from uptake of ethylamine and perchlorate, assuming the ions taken up were free in solution inside the vesicles, was 23 to 24 kJ/mol of H+ (240-250 mV) during respiration in the absence of ATP. The ratio of the free energy of ATP synthesis (AGAT~) to A,iH+ was 2.2 to 2.3 during oxidative phosphorylation and only slightly higher during ATP hydrolysis indicating that the H+-translocating ATPase is close to equilibrium under both conditions. The nonintegral ratio suggests there is a systematic error in the measurement of