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Characterization of the atractyloside-sensitive adenine nucleotide transport system in rat liver mitochondria.

H. Winkler,F. Bygrave,A. Lehninger

Published 1968 in Journal of Biological Chemistry

ABSTRACT

Abstract By means of an improved Millipore filtration technique, a very rapid, atractyloside-sensitive exchange of adenine nucleotides between intact rat liver mitochondria and the medium has been studied. The uptake of ADP or ATP has a rapid phase with a half-time of the order of 6 sec at 0°, followed by a slower phase resulting in a plateau level of 8 to 10 nmoles of exchangeable nucleotide per mg of mitochondrial protein. The uptake system has a very high affinity for ADP (Km l 30 µm) and is highly specific for adenine nucleotides (ADP g ATP >> AMP). Other purine and pyrimidine nucleotides are inactive and, in addition, fail to inhibit the uptake of 14C-ADP. The uptake of adenine nucleotide occurs by an obligatory exchange diffusion process, i.e. the entrance of 1 molecule of external nucleotide was found to be coupled to the exit of 1 molecule of internal nucleotide, resulting in a constant internal pool size, even when external ADP concentration was varied over a wide range. Adenine nucleotide uptake is insensitive to thiol reagents and to uncouplers and inhibitors of oxidative phosphorylation; however, it may be completely inhibited by very low levels of atractyloside (Ki = 1 µm). Maximum atractyloside inhibition requires Mg++ when ADP is the external nucleotide but not when ATP is present. However, Mg++ is not required for the uptake of either ADP or ATP in the absence of atractyloside. The internal pool of labeled nucleotides consists of a mixture of ATP, ADP, and AMP; its composition is about the same after equilibration with either 14C-ADP or 14C-ATP. Since rat liver mitochondria stripped of their outer membranes showed atractyloside-sensitive adenine nucleotide uptake very similar to that of the intact mitochondria, it is concluded that the atractyloside-sensitive sites are in the inner membrane. The data support the general conclusion that the nucleotide exchange process is brought about by a catalytic carrier in the membrane acting between external and internal nucleotide pools, rather than by stoichiometric binding of the nucleotide to a large number of mitochondrial sites.

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