Macromolecules increase the channelling of ADP from externally associated hexokinase to the matrix of mitochondria

Macromolecules can restore the morphological changes in the outer mitochondrial compartment that occur upon isolation of the organelle. They decrease the volume of the intermembrane space and increase the number of intermembrane contact sites. In this study, we investigated the effects of macromolecules on one of the processes occurring in the mitochondrial outer compartment and for which the native structure might be important, i.e. the ADP supply from outer-membrane-bound hexokinase-I to oxidative phosphorylation. With the use of a reconstituted system in which rat liver mitochondria and extramitochondrial pyruvate kinase compete for ADP generated by hexokinase, it was shown that (a) part of the ADP generated by mitochondrially associated hexokinase is not accessible to pyruvate kinase and is channeled into the mitochondrion, (b) in the presence of 10% (mass/vol.) macromolecules (i.e. dextran M20 or BSA) the pyruvate kinase inaccessible fraction increases from 19% to 31 % of the ADP produced by hexokinase, (c) the ADP channeling is a characteristic property of bound hexokinase, and (d) the increased channeling induced by macromolecules can neither be explained by direct effects of these macromolecules on the basic respiratory properties of rat liver mitochondria, nor by direct effects on the kinetic properties of hexokinase-I. ATP and ADP determinations were performed in hexokinase/mitochondria incubation mixtures in the presence of macromolecules. These determinations showed that an important consequence of the channeling capacity of bound hexokinase is that lower extramitochondrial ADP levels and consequently higher extramitochondrial ATP/ADP ratios are maintained than when hexokinase is not bound. The experimental data demonstrate that the ADP channeling activity associated with bound hexokinase leads to the formation of two ADP concentration gradients, one across the outer membrane and one between bound hexokinase and the bulk phase