Kinetic, binding and ultrastructural properties of the beef heart adenine nucleotide carrier protein after incorporation into phospholipid vesicles

Abstract

1. 1. ADP/ATP transport has been reconstituted by incorporation of the purified carrier protein in liposomes filled with ATP. The transport was assayed by uptake of [ 14C]ADP into the liposomes, and by release of ATP as determined by a luminescence technique. [ 14C]ADP uptake was strictly dependent on internal ATP. 2. 2. The simplest phospholipid system capable of yielding high rates of ADP/ATP transport was a mixture of phosphatidylethanolamine and cardiolipin (92 : 8, w/w). 3. 3. ADP/ATP transport in the reconstituted system proceeded by exchange-diffusion with a 1 1 stoichiometry. The specificity for ADP and ATP was absolute. The capacity and the rate of exchange depended on the concentration of ATP present in liposomes. The rate of transport at 20°C, at 20 mM internal ATP, routinely ranged between 300 and 1000 nmol of nucleotide exchanged per min/mg of added carrier protein. The apparent K m value for external ADP was around 10 μM. 4. 4. The ADP/ATP exchange in the reconstituted system was rather stable to ageing. It dropped by only 20% after 1 day of ageing at 20°C. Divalent cations (Mg 2+, Mn 2+, Ca 2+) at concentrations higher than 1 to 2 mM had a deleterious effect on ADP/ATP transport, concomitant with the release of internal ATP and accumulation of multilamellar vesicles. 5. 5. Atractyloside behaved as a competitive inhibitor and carboxyatractyloside as a non-competitive inhibitor. Bongkrekic acid required a slightly acidic pH to be inhibitory. The data concerning atractyloside, carboxyatractyloside and bongkrekic acid were similar to those obtained with whole mitochondria, suggesting that the carrier protein in liposomes has the same asymmetrical arrangement as in mitochondria. 6. 6. The percentage of competent carrier protein in liposomes was calculated from dose-response data concerning the inhibition of ADP/ATP transport by atractyloside or carboxyatractyloside, and from the amount of bound [ 3H]-atractyloside removable by ADP. By both methods, 3 to 6% of the added carrier protein was found to be competent in ADP/ATP transport, based on the assumption that the binding of one atractyloside or carboxyatractyloside molecule per 30 000 molecular weight carrier unit results in complete inhibition of transport. 7. 7. Freeze-fracture electron microscopy showed that the ADP/ATP carrier protein-lipid preparations are formed by small vesicles, most of which give rise to smooth fracture faces (probably pure lipid vesicles). Only a small percentage of the vesicles (2 to 4% depending on the amount of carrier protein added) were clearly particulated. About 90% of the particulated vesicles showed no more than 2 particles per vesicle and only 5% more than 5 particles per vesicle. The distribution of the particles between convex and concave fracture faces was asymmetric; about 2 3 of the protein molecules were anchored at the external surface of the vesicles and only 1 3 at the internal one. This asymmetric distribution was not significantly modified after the addition of carboxyatractyloside but changed drastically upon addition of bongkrekic acid, leading to an increased percentage of protein molecules anchored at the internal surface of the vesicles. 8. 8. The above experimental data suggest that the ADP/ATP carrier protein is able to move by translation across the phospholipid membrane. The data are interpreted by assuming that the carrier is stabilized in a conformation more exposed to the inside upon bongkrekic acid binding, and to the outside upon (carboxy)atractyloside binding. A similar translational motion could be involved in ADP/ATP transport.