Intramitochondrial and extramitochondrial free calcium ion concentrations of suspensions of heart mitochondria with very low, plausibly physiological, contents of total calcium.

The 2-oxoglutarate dehydrogenase of intact rat heart mitochondria is activated by Ca2+, with 50% activation at approximately 0.5 nmol of total Ca/mg of mitochondrial protein, in the presence of Pi and Mg2+. Mitochondrial Ca contents in excess of 2 nmol/mg of protein result in 100% activation of the enzyme. Investigation of Ca2+ release from the mitochondria using the metallochromic indicator Arsenazo III defines a S0.5 of 5.4 +/- 0.4 nmol of Ca/mg of protein, when the endogenous Ca content of the mitochondria is progressively depleted with EGTA, prior to the initiation of the release process being studied. The subsequent determination of matrix free Ca2+ concentration by the "null-point" technique has allowed expression of these results in terms of free concentration rather than Ca content, with an activity coefficient of approximately 0.001 for matrix Ca2+. From the above, Ca2+ efflux from heart mitochondria is not saturated at the mitochondrial Ca contents or Ca2+ concentrations which give effective regulation of dehydrogenase activity. A consequence is that heart mitochondria do not buffer the pCa of the extramitochondrial medium at these Ca contents (less than 2 nmol/mg of protein), and this is shown in direct measurements of extramitochondrial pCa. This is taken to question the physiological significance of mitochondrial buffering of cytosolic free Ca2+ in normal heart.