JBC, Vol. 251, Issue 4, 968-974, Feb, 1976
Stoichiometry of H+ ejection during respiration-dependent accumulation of Ca2+ by rat liver mitochondria
M. D. Brand, C. H. Chen and A. L. Lehninger
We have investigated the energy-dependent uptake of Ca2+ by rat liver
mitochondria with succinate as respiratory substrate with rotenone added to
block NAD-linked electron transport. In the presence of 3-hydroxybutyric or
other permeant monocarboxylic acids Ca2+ was taken up to extents
approaching those seen in the presence of phosphate. The quantitative
relationship between cation and anion uptake was determined from the slope
of a plot of 3-hydroxybutyrate uptake against Ca2+ uptake, a method which
allowed determination of the stoichiometry without requiring ambiguous
corrections for early nonenergized or nonstoichiometric binding events.
This procedure showed that 2 molecules of 3-hydroxtbutyrate were
accumulated with each Ca2+ ion. Under these conditions close to 2 Ca2+ ions
and 4 molecules of 3-hydroxybutyrate were accumulated per pair of electrons
per energy-conserving site of the respiratory chain. Since
3-hydroxybutyrate must be protonated to pass the membrane as the
undissociated free acid, it is concluded that 4 protons were ejected (and
subsequently reabsorbed) per pair of electrons per energy-conserving site,
in contrast to the value 2.0 postulated by the chemiosmotic hypothesis.
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