Factors affecting the translocation of oxaloacetate and L-malate into rat liver mitochondria.
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 K(m) values were 40mum 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 K(i) 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 50mum-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.
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000814:Aniline Compounds / Q000494:pharmacology; D000818:Animals; D001205:Ascorbic Acid / Q000494:pharmacology; D003864:Depression, Chemical; D066298:In Vitro Techniques; D007700:Kinetics; D008293:Malates / Q000378:metabolism*; D008930:Mitochondria, Liver / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D010071:Oxaloacetates / Q000378:metabolism*; D010085:Oxidative Phosphorylation; D051381:Rats; D013053:Spectrophotometry; D013268:Stimulation, Chemical; D013696:Temperature
D000814:Aniline Compounds; D008293:Malates; D010071:Oxaloacetates; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D001205:Ascorbic Acid