KINETICS OF NUCLEOTIDE TRANSPORT IN RAT-HEART MITOCHONDRIA STUDIED BY A RAPID FILTRATION TECHNIQUE

A rapid filtration technique has been used to measure at room temperature the kinetics of ADP and ATP transport in rat heart mitochondria in the millisecond time range. Transport was stopped by cessation of the nucleotide supply, without the use of a transport inhibitor, thus avoiding any quenching delay. The mitochondria were preincubated for 30 s either in isotonic KCl containing succinate, MgCl2, and Pi (medium P) or in isotonic KCl supplemented only with EDTA and Tris (medium K); they were referred to as energized and resting mitochondria, respectively. The kinetics of [14C]ADP transport in energized mitochondria were apparently monophasic. The plateau value for [14C]ADP uptake reached 4-5 nmol of nucleotide·(mg of protein)−1. Vmax values for [14C]ADP transport of 400–450 nmol exchanged-min−1·(mg of protein)−1 with Km values of the order of 13–15 μM were calculated, consistent with rates of phosphorylation in the presence of succinate of 320–400 nmol of ATP formed·min−1·(mg of protein)−1. The rate of transport of [14C]ATP in energized mitochondria was 5–10 times lower than that of [14C]ADP. Upon uncoupling, the rate of [14C]ATP uptake was enhanced, and that of [14C]ADP uptake was decreased. However, the two rates did not equalize, indicating that transport was not exclusively electrogenic. Transport of [14C]ADP and [l4C]ATP by resting mitochondria followed biphasic kinetics. These consisted of a rapid nucleotide uptake of about 350 nmol·min−1·(mg of protein)−1, lasting for about 1 s, up to 1.0–1.2 nmol of [14C]nucleotide taken up·(mg of protein)−1, followed by a slow phase leading to a plateau value of 4–5 nmol of [14C]nucleotide·(mg of protein)−1, which was attained in 1 min. Depletion of nucleotides in resting mitochondria resulted in a greater decrease in the extent of the slow phase than of the rapid one. In addition, about half of the nucleotides taken up at the end of the rapid phase were not discharged into the medium upon addition of carboxyatractyloside. This suggested that matricial nucleotides are compartmentalized in two pools which are exchangeable at different rates with external nucleotides. © 1990, American Chemical Society. All rights reserved.