Kinetics of electrogenic transport by the ADP/ATP carrier.
The electrogenic transport of ATP and ADP by the mitochondrial ADP/ATP carrier (AAC) was investigated by recording transient currents with two different techniques for performing concentration jump experiments: 1) the fast fluid injection method: AAC-containing proteoliposomes were adsorbed to a solid supported membrane (SSM), and the carrier was activated via ATP or ADP concentration jumps. 2) BLM (black lipid membrane) technique: proteoliposomes were adsorbed to a planar lipid bilayer, while the carrier was activated via the photolysis of caged ATP or caged ADP with a UV laser pulse. Two transport modes of the AAC were investigated, ATP(ex)-0(in) and ADP(ex)-0(in). Liposomes not loaded with nucleotides allowed half-cycles of the ADP/ATP exchange to be studied. Under these conditions the AAC transports ADP and ATP electrogenically. Mg(2+) inhibits the nucleotide transport, and the specific inhibitors carboxyatractylate (CAT) and bongkrekate (BKA) prevent the binding of the substrate. The evaluation of the transient currents yielded rate constants of 160 s(-1) for ATP and >/=400 s(-1) for ADP translocation. The function of the carrier is approximately symmetrical, i.e., the kinetic properties are similar in the inside-out and right-side-out orientations. The assumption from previous investigations, that the deprotonated nucleotides are exclusively transported by the AAC, is supported by further experimental evidence. In addition, caged ATP and caged ADP bind to the carrier with similar affinities as the free nucleotides. An inhibitory effect of anions (200-300 mM) was observed, which can be explained as a competitive effect at the binding site. The results are summarized in a transport model.
Gropp
T
T
Max-Planck-Institut für Biophysik, 60596 Frankfurt, Germany.
Brustovetsky
N
N
Klingenberg
M
M
Müller
V
V
Fendler
K
K
Bamberg
E
E
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D000818:Animals; D001693:Biological Transport, Active / Q000187:drug effects; D055592:Biophysical Phenomena; D001703:Biophysics; D002417:Cattle; D004563:Electrochemistry; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007700:Kinetics; D008081:Liposomes; D008274:Magnesium / Q000494:pharmacology; D008929:Mitochondria, Heart / Q000201:enzymology; D000226:Mitochondrial ADP, ATP Translocases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D008954:Models, Biological; D013816:Thermodynamics
D004791:Enzyme Inhibitors; D008081:Liposomes; D000244:Adenosine Diphosphate; C029273:P(3)-1-(2-nitro)phenylethyladenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D000226:Mitochondrial ADP, ATP Translocases; D008274:Magnesium