Reaction mechanism of the reconstituted oxoglutarate carrier from bovine heart mitochondria.
The transport mechanism of the reconstituted oxoglutarate carrier, purified from bovine heart mitochondria, was studied kinetically. A complete set of half-saturation constants (Km) was established for the two different substrates oxoglutarate and malate on both the external and the internal sides of the membrane. The internal affinities for oxoglutarate (Km 0.17 mM) and malate (Km 0.7 mM) were higher than the corresponding external affinities (Km 0.3 mM and 1.4 mM, respectively). The exclusive presence of a single transport affinity for each substrate on one side of the membrane indicated a unidirectional insertion of the oxoglutarate carrier into the liposomal membrane. The Km values and also the maximum exchange rates (8-11 mumol.min-1.mg protein-1) for oxoglutarate and malate were independent of the nature of the counter substrate on the other side of the membrane. Under these defined conditions we analyzed the antiport mechanism in two-reactant initial velocity studies varying both the internal and external substrate concentrations. From the kinetic patterns obtained, a sequential type of mechanism became evident, implying that one internal and one external substrate molecule form a ternary complex with the carrier before transport occurs. A quantitative analysis of substrate interaction with the unloaded or single-substrate-occupied carrier revealed that rapid-equilibrium random conditions were fulfilled, characterized by a fast and independent binding of internal and external substrate. This kinetic mechanism agrees with previous results obtained in intact mitochondria. Considering also the data available for other mitochondrial carriers, a common kinetic mechanism (sequential type) for this carrier family is suggested.
Indiveri
C
C
Department of Pharmaco-Biology, University of Bari, Italy.
Dierks
T
T
Krämer
R
R
Palmieri
F
F
D000818:Animals; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism*; D002417:Cattle; D007425:Intracellular Membranes / Q000378:metabolism; D007656:Ketoglutaric Acids / Q000378:metabolism*; D007700:Kinetics; D008081:Liposomes; D008293:Malates / Q000378:metabolism; D026901:Membrane Transport Proteins; D008929:Mitochondria, Heart / Q000378:metabolism*; D011510:Proteolipids / Q000378:metabolism; D013379:Substrate Specificity
D002352:Carrier Proteins; D007656:Ketoglutaric Acids; D008081:Liposomes; D008293:Malates; D026901:Membrane Transport Proteins; D011510:Proteolipids; C021723:oxoglutarate translocator; C020485:proteoliposomes