The number of H+ ejected during passage of 2e- through each energy conserving site of the mitochondrial respiratory chain (the H+/site ratio) was measured in three ways. In each case transmembrane movements of endogenous phosphate were minimized. Measurement of the uptake of weak acids during loading of mitochondria with Ca2+ demonstrated that 2.0 weak acid anions were accumulated per Ca2+ ion. Since 1.7 to 2.0 Ca2+ ions were taken up per site, these data correspond to an H+/site ratio of 3.5 to 4.0. More direct measurement of H+ ejection using the oxygen pulse technique demonstrated that the H+/site ratio was 3.0. In these experiments phosphate movements were prevented by addition of N ethylmaleimide to inhibit phosphate hydroxide antiport, by washing the mitochondria to remove endogenous phosphate, or by working at 5°C to reduce the rate of phosphate transport. When phosphate movements were allowed, H+/site ratios of 2.0 were observed. Measurement of the initial steady rates of oxygen consumption and H+ ejection following addition of substrate to aerobic, substrate limited mitochondria yielded H+/site ratios of 2.0, which were elevated to 4.0 when phosphate transport was prevented as described above. Previous determinations of the H+/site ratio were thus underestimates due to the unrecognized movements of endogenous phosphate; the results show that the H+/site ratio is at least 3.0 and may be as high as 4.0.
|Original language||English (US)|
|Number of pages||8|
|Journal||Journal of Cellular Physiology|
|State||Published - 1976|
ASJC Scopus subject areas
- Cell Biology
- Clinical Biochemistry