Use of Carboxyatractylate and Tight-Binding Inhibitor Theory to Determine the Concentration of Functional Mitochondrial Adenine Nucleotide Translocators in a Reconstituted System

Abstract

The adenine nucleotide translocator (AdNT) has been isolated from rat heart mitochondria and reconstituted into liposomes containing ATP. Translocator activity was determined using a coupled enzyme system to measure the ADP-induced efflux of ATP from the liposomes. In order to determine specific activity, the number of functional translocators must also he known. Carboxyatractylate (CAT) is a highly selective inhibitor of the AdNT, with Ki < 10 nM, a value sufficiently low relative to the concentration of protein in transport assays to suggest the use of tight-binding inhibitor theory to quantify functional translocators. Ackermann-Potter plots of velocity vs proteoliposome concentration at several different CAT concentrations were used both to demonstrate the occurrence of tight-binding inhibition and to determine the concentration of AdNT catalytic sites in the native orientation. The results obtained agreed well with earlier reports based on [14C]CAT binding; functionally reconstituted AdNT represented 5-10% of the protein added to the system. Specific activities were ca. 7-10 μmol/min mg depending on the lipid composition of the liposomes. Unincorporated protein did not appreciably affect the measurements. This methodology should be readily applicable to any reconstituted systems for which high-affinity inhibitors which bind only to active protein are known.