| Abstract: | 1. The distribution of labeled and unlabeled adenine-nucleotides inside and outside mitochondria was followed after addition of 14C]ADP to rat liver mitochondria. Two types of mitochondria were used: 1, respiring mitochondria which were carrying out oxidative phosphorylation and which had been replenished in ATP by incubation in a medium supplemented with succinate and phosphate; 2, non-respiring mitochondria which had been partially depleted of ATP by incubation in a medium supplemented with rotenone and phosphate. During the first minute following addition of 14C]ADP to the respiring mitochondria, the pre-existing intramitochondrial (internal) 12C]ATP was released into the medium and replaced by newly synthesized 14C]ATP. No 14C]ADP accumulated in the mitochondria. It is suggested that extramitochondrial (external) ADP entering respiring mitochondria in exchange for internal ATP is phosphorylated to ATP before its complete release in the matrix space. In non-respiring mitochondria, the entry of 14C]ADP into the mitochondria was accompanied by the appearance in the external space of 12C]ADP and 12C]ATP, with a marked predominance of 12C]ADP. Thus in non-respiring mitochondria, the residual internal ATP is dephosphorylated to ADP in the inner membrane before being released outside the mitochondria. 2. When mitochondria were incubated with glutamate, ADP and 32P]phosphate, the 32P]ATP which accumulated in the matrix space became rapidly labeled in both the P gamma and P beta groups of the ATP, due to the presence of a transphosphorylation system in the mitochondrial matrix. The 32P]ATP which accumulated outside the mitochondria was also labeled in the P beta group, although less rapidly than the internal ATP. Our data show that a large fraction (75-80%) of the ATP produced by phosphorylation of added ADP within the inner mitochondrial membrane is released into the matrix space before being transported out from the mitochondria; only a small part (20-25%) is released directly outside the mitochondria without penetrating the matrix space. 3. In respiring and phosphorylating mitochondria, the value of the Km of the ADP-carrier for external ADP was 2-4 times lower than its value in non-respiring and non-phosphorylating mitochondria. 4. The above experimental data are discussed with reference to the topological and functional relationships between the ADP-carrier and the oxidative phosphorylation complex in the inner mitochondrial membrane. They strongly suggest that the ADP-carrier comes to the close neighbourhood of the ATP synthetase on the matrix side of the inner membrane. |
