The impermeability of the mitochondrial inner membrane to the chelator ethanedioxybis(ethylamine)tetra-acetic acid permits discrimination between Ca2+ which has been transported to the internal (matrix) phase and Ca2+ which binds to the external surfaces of the mitochondrion. With this technique, it is shown that `energy-independent high-affinity' binding is a measure of carrier-mediated active Ca2+ transport in respiration-inhibited mitochondria; the carrier also transports Ca2+ to the internal phase after treatment with carbonyl cyanide m-chlorophenylhydrazone, but in this case the active-transport component is inhibited. The Ca2+-binding sites associated with the external membrane surfaces are similar in concentration and affinity for both inhibited and uncoupled mitochondria; it was not possible to measure external Ca2+ binding which could be identified as carrier specific. The results are discussed in relation to the mechanism of mitochondrial Ca2+ transport, and to previous studies of energy-independent Ca2+ binding.
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