Relations between extramitochondrial and intramitochondrial adenine nucleotide systems

The control of oxidative phosphorylation by the extramitochondrial $\text{ATP}\text{]}\text{ADP}\text{]}$ ratio and P_i] was investigated by incubations of isolated mitochondria with an ADP regenerating system and by a new perifusion technique using glass filters for immobilization of mitochondria. With mitochondria from different sources oxidizing different substrates and with both techniques, similar results were obtained. Changes of the extramitochondrial $\text{ATP}\text{]}\text{ADP}\text{]}$ ratio from about 100 to 5 transfer mitochondria from the resting state (state 4) to the fully active state (state 3). The importance of the adenine nucleotide translocator in this transition was demonstrated by the influence of its specific inhibitor carboxyatractyloside. The sensitivity to the inhibitor was more pronounced in states with high $\text{ATP}\text{]}\text{ADP}\text{]}$ ratios than in the fully active state. In the hexokinase-glucose system the action of the inhibitor caused a transition to a new steady state, where a decreased $\text{ATP}\text{]}\text{ADP}\text{]}$ ratio overcomes the inhibition. Thus, a partial inhibition of the translocator shifted the control characteristics to lower $\text{ATP}\text{]}\text{ADP}\text{]}$ ratios. When the concentration of inorganic phosphate was decreased, the main effect was a reduction of the maximum rate of oxidative phosphorylation (i.e., in state 3), whereas the $\text{ATP}\text{]}\text{ADP}\text{]}$ sensitive range was not altered. This effect is caused by changes in the intramitochondrial phosphorylation potential. Furthermore, this indicates that the kinetic properties of the adenine nucleotide translocator prevent a simple equilibration of the phosphorylation potential across the inner membrane. This is also demonstrated by the fact that the extramitochondrial formation of glucose-6-phosphate and the intramitochondrial synthesis of citrulline compete for ATP.