Adenine Nucleotide Translocation of Mitochondria: 1. Specificity and Control

Abstract

The adenine nucleotide translocation in mitochondria has previously been established as an exchange between exogenous and endogenous adenine nucleotides across the inner membrane. The specificity and the control of the exchange are examined with the following major results: The adenine nucleotide translocation is relatively specific for exogenous ADP and ATP, AMP being nearly inactive. Among other nucleotides tested, only dADP and dATP exchange with a noticeable activity. In the controlled state ADP exchanges 2–4 times faster than ATP. If simultaneously added, ADP and ATP compete for the exchange, with ADP being about tenfold more active than ATP. The specificity of the exit of adenine nucleotides in the exchange is similar to the specificity of the entrance with the difference that ADP and ATP are released with equal activity in proportion to their intramitochondrial content. AMP is released only after a slow conversion to ADP. Therefore the short time exchange is limited by the endogenous content of ADP plus ATP. The exchange is influenced by the metabolic state of the mitochondria. The ATP exchange is more variable than the ADP exchange. Two effects are elucidated: (a) the influence of the metabolic state on the relative content of AMP which inhibits both the ADP and ATP exchange (b) the coupling of the energy transfer system which inhibits only the ATP exchange. An example for case (a) is the inhibition of the ADP and ATP exchange by arsenate and an example for case (b) is the strong increase of the ATP exchange on uncoupling. The following effects are relevant to the mechanism of the control of the exchange by ATP. The stimulation of the ATP exchange by uncoupler has the same concentration dependence as the uncoupling of oxidative phosphorylation (Km [CCP] = 0.08 μM, where CCP = carbonyl‐cyanide‐phenylhydrazone). Oligomycin does not abolish the uncoupler effect on the ATP exchange. “Endogenous uncoupling” on aging of mitochondria also stimulates the ATP exchange. Valinomycin plus K+ only slightly stimulate the ATP exchange. Anaerobiosis stimulates the ATP exchange to a smaller extent than uncoupling. In competition with ADP the effects of energy transfer on ATP exchange are more strongly revealed. On uncoupling the more than tenfold preference for ADP is fully abolished. It is concluded that basically the exchange for ADP and ATP has equal specificity in forward and reverse reaction. In the controlled state a superimposed force makes the specificity asymmetric and inhibits the entrance of ATP. This control of the ATP exchange is concluded to be based on the anionic character of the adenine nucleotides. Thus the ATP 4‐ex–ADP 3‐in exchange is inhibited unless the charge difference is compensated for by an uncoupler stimulated H+ movement across the membrane. Furthermore an electric potential gradient appears to be effective in the controlled state which is abolished on uncoupling.