Non-phosphorylated intermediates in oxidative phosphorylation

Abstract

1. 1. This paper is concerned with the study of the formation of non-phosphorylated intermediates in digitonin particles prepared from rat-liver mitochondria. 2. 2. Digitonin particles were preincubated with either β-hydroxybutyrate, sucinate or ferrocytochrome c for a short time at 20°. After cooling the preincubated mixture was incubated at 1° with 32P i and ADP. Under these conditions, the time course of [ 32P]ATP synthesis is represented by a biphasic curve. The initial portion corresponds to a high rate of [ 32P]ATP synthesis whereas the second portion corresponds to a slower rate. 3. 3. The rapid initial [ 32P]ATP synthesis is maximal for pH values ranging from 6.5 to 7.3. It is increased by compounds which increase the efficiency of oxidative phosphorylation in digitonin particles such as EDTA and serum albumin and is inhibited by inhibitors of oxidative phosphorylation such as 2,4-dinitrophenol or azide. It is delayed or considerably depressed when unlabeled P i is added during the preincubation time prior to the addition of 32P i and ADP but is not affected by atractylate, a compound which is capable of specifically inhibiting the ATP-P i exchange in digitonin particles without interfering with the ATP synthesis linked to respiration. Finally it is enhanced by low concentrations of oligomycin which at 1° have little effect on the rate and the yield of oxidative phosphorylation. 4. 4. The effect of 32P i concentration on the initial rate of [ 32P]ATP synthesis was determined: K m = 1.7·10 −3 M. 5. 5. The experimental findings have been interpreted as follows: non-phosphorylated intermediates accumulate in digitonin particles during the oxidation of β-hydroxybutyrate, succinate or ferrocytochrome c. These intermediates are discharged upon addition of P i and ADP, the net result of the reaction being the synthesis of ATP.