Elucidation of a Carrier Site for Adenine Nucleotide Translocation in Mitochondria with the Help of Atractyloside

Abstract

Previously, the adenine translocation through the mitochondrial membrane has been shown to be mediated by a carrier system which catalyses a compulsory exchange between exogenous and endogenous adenine nucleotides. So far, the study of the adenine nucleotide translocation has been mainly concerned with a description of the overall process (specificity, kinetics, temperature dependence, regulation, etc.) [1–3]. For further understanding of the mechanism it is of interest to determine whether one can detect binding sites for adenine nucleotides and, if so, to measure the number of sites which can be attributed to the carrier per g of mitochondrial protein. As explained in detail by Heldt [4], atractyloside has been shown to be a highly specific and effective inhibitor of adenine nucleotide translocation. It can be assumed that atractyloside inhibits by way of competitive binding to the adenine nucleotide carrier site. In view of its high affinity, one type of approach for measuring the carrier sites would be to study atractyloside binding. An alternative method would be to determine the binding of adenine nucleotides (ADP or ATP) and to differentiate the specific from the unspecific binding by displacement of the adenine nucleotides with atractyloside. This approach is made possible by the unusually high affinity of the adenine nucleotide exchange for ADP or ATP. It is complicated by the fact that adenine nucleotides incorporated into the intramitochondrial space by exchange will be indistinguishable from adenine nucleotides bound to the carrier, unless some means of identifying the bound and exchanged fractions is employed.