1. The rates of translocation of oxaloacetate and l-malate into rat liver mitochondria were measured by a direct spectrophotometric assay. 2. Penetration obeyed Michaelis–Menten kinetics, and apparent Km values were 40μm for oxaloacetate and 0·13mm for l-malate. 3. Arrhenius plots of the temperature-dependence of rates of penetration gave activation energies of +10kcal./mole for oxaloacetate and +8kcal./mole for l-malate. 4. The translocation of both oxaloacetate and l-malate was competitively inhibited by d-malate, succinate, malonate, meso-tartrate, maleate and citraconate. The Ki values of these inhibitors were similar for the penetration of both oxaloacetate and l-malate. 5. Rates of penetration were stimulated by NNN′N′-tetramethyl-p-phenylenediamine dihydrochloride plus ascorbate under aerobic conditions or by ATP under anaerobic conditions. 6. The energy-dependent stimulation of translocation was abolished by uncouplers of oxidative phosphorylation. Oligomycin A, aurovertin, octyl-guanidine and atractyloside prevented the stimulation by ATP, but did not inhibit the stimulation by NNN′N′-tetramethyl-p-phenylenediamine dihydrochloride plus ascorbate. 7. Mitochondria prepared in the presence of ethylene-dioxybis(ethyleneamino)tetra-acetic acid did not exhibit the energy-dependent translocation, but this could be restored by the addition of 50μm-calcium chloride. 8. Valinomycin or gramicidin plus potassium chloride enhanced the energy-dependent translocation of oxaloacetate and l-malate. 9. Addition of oxaloacetate stimulated the adenosine triphosphatase activity of the mitochondria, and the ratio of ‘extra’ oxaloacetate translocation to ‘extra’ adenosine triphosphatase activity was 1·6:1. 10. Possible mechanisms for the energy-dependent entry of oxaloacetate and l-malate into mitochondria are discussed in relation to the above results.
Environmental CO2 Stimulation of Mitochondrial Adenosine Triphosphatase Activity
