The adenine-nucleotide exchange in submitochondrial (sonic) particles.

Abstract

Transport of adenine nucleotides through the inner mitochondrial membrane is investigated with submitochondrial (sonic) particles from beef heart mitochondria. Significant transport of nucleotides is observed only when the particles are loaded with adenine nucleotides during sonication. The nucleotide transport is characterized as follows. 1 Sonic particles can be loaded during sonication in the presence of 20 mM ADP or ATP with up to 18 μmol/g protein, of a mixture of ATP, ADP and AMP. 2 The loaded particles incorporate labelled ADP or ATP up to 6 μmol/g protein relatively slowly (10°C, t1/2= 20 s). 3 By measuring and labelling the endogenous nucleotide pool, the nucleotide transport was identified as a counter-exchange. 4 In contrast to mitochondria the inhibition by bongkrekate is rapid, independent of pH and preincubation with ADP. This indicates that bongkrekate inhibits the carrier from the outside. 5 The exchange is inhibited by bongkrekate but not by atractylate unless the particles are derived from mitochondria preloaded with atractylate. This indicates asymmetry of the translocation with respect to its orientation to the membrane side: When oriented to the ‘m-side’ (outside in submitochondrial particles) the affinity is high for bongkrekate and low for atractylate and in the ‘side’ it is high for atractylate and low for bongkrekate. These results are in accordance with the predictions made from studies on mitochondria. In both states it has high affinity for palmityl-CoA. 6 The translocation rate in sonic particles is about 18 μmol x min−1 x g protein-1 at 20°C and amounts to 6% of the rate in the parent mitochondria. Translocation rate has a high temperature dependence (Q10= 3.5) with Ea= 24 kcal (100 kJ) in the range between 0 and 24°C and between 24°C and 35°C, Ea= 4 kcal (17 kJ) ‘break’ in the temperature dependence is at about 25°C. 7 Specificity of the nucleotide exchange is even higher than that of mitochondria. Exchange with dADP and AMP-P(NH)P reaches only about 3% of the activity with ADP. The ADP uptake rate is about 30% faster than that of ATP. There is little influence of uncoupling by carbonylcyanide p-trifluoromethoxyphenylhydrazone in contrast to mitochondria. 8 The exchange is not inhibited by N-ethylmaleimide unless N-ethylmaleimide is added prior to the sonication. The results demonstrate that in sonic particles the nucleotide can be transported similar to that in mitochondria, however, with strongly reduced activity. The exposure of the m-side in the particles gives important results about the asymmetry of the carrier properties.