Conformational Changes and Possible Structure of the Oxoglutarate Translocator of Rat‐Heart Mitochondria Revealed by the Kinetic Study of Malate and Oxoglutarate Uptake

Abstract

+ oxoglutarate),,, : malatein catalysed by the oxoglutarate carrier of rat-heart mitochondria have been studied under conditions where internal and external substrates may be varied.It is shown that contrary to external oxoglutarate which induces a conformational change of the translocator subunit to which it binds, external malate does not induce conformational changes during its binding and is a Michaelian substrate.The study of the effect of external malate on the rate of oxoglutarate uptake shows that external malate and external oxoglutarate are competitive.External oxoglutarate affects the catalytic rate constant of malate uptake in a modulated way. After substrate binding, the exchange reaction between an external dicarboxylate and an internal dicarboxylate is accompanied by conformational changes. The particular form of the rate equation strongly suggests that during a first step the external substrate bound to an external binding subunit at the external surface of the membrane, and the internal substrate bound to an internal binding subunit at the internal surface of the membrane, are transferred to a catalytic subunit (channel?) deeper in the membrane. Two models, one with a single channel, and the other with several associated channels, are proposed.It is demonstrated that a binding subunit which has transferred its substrate to a catalytic subunit is left in a conformation which does not depend on the substrate that has 'passed through it'. It is also demonstrated that all the catalytic subunits are identical. These theoretical dcductions allow a simple description of the complicated effect that external oxoglutarate has on the rate of malate uptake.The fact that all the external binding subunits arc equivalent regarding external malate binding and that all the catalytic subunits are identical support the view that the mitochondria1 preparation contains a single species of oxoglutarate translocator and not an isozymic mixture.The oxoglutarate translocator present in rat-heart mitochondria [I] is located in the inner membrane and performs one-to-one exchanges between an external and an internal dicarboxylate anion as shown explicitly in [2].The oxoglutarate translocator is a component of the malate-aspartate shuttle which transfers reducing equivalents produced in the cytosol to the respiratory chain located in the mitochondria. This shuttle is not only the main transfer device in heart but is also strictly necessary in working heart preparations metabolizing glucose, since aminooxyacetate, an inhibitor of the transamination steps of the shuttle, causes leftventricular failure, decreases myocardial respiration and increases lactate production [3]. By recycling the glycolytic NADH, the malate-aspartate shuttle thus protects pyruvate from reduction allowing its vital oxidation by the Krebs cycle.The glycolytic activity in the sarcoplasm and the oxidative phosphorylation in the mitochondria are linked by transmcmbrane translocators : the pyruvate, adenine-nucleotide, oxogluAhhreviatlon.s. Mal, malate; OG...

Reaction mechanism of the reconstituted oxoglutarate carrier from bovine heart mitochondria

“…In previous studies on the kinetics of the oxoglutarate translocator carried out in intact mitochondria [2,3,6,8,91, the K, values for external substrates were reported to be significantly lower (cf. Table 1) than those shown above for either side of the liposomal membrane.…”

“…We thereby succeeded in obtaining all kinetic constants necessary to describe the two different sides of the reconstituted oxoglutarate carrier (Table 1) showing medium-affinity bind- ing at the inside ( K , 0.2 mM and 0.7 mM for oxoglutarate and malate, respectively) and medium-to-low-affinity binding at the outside of the liposomes ( K , 0.3 mM and 1.4 mM). When comparing the kinetic data obtained here with those from detailed studies in intact mitochondria [2][3][4][5][6][7][8][9]251, it becomes obvious that the situation in the proteoliposomes is definitely less complicated. We did not observe cooperativity phenomena and, due to the absence of other metabolites, no possible allosteric influences had to be considered (see above).…”

“…On the basis of the most relevant K, found in rat heart mitochondria, i.e. the K, for external malate which was the only Michaelian substrate a t the cytosolic side [6], one could speculate on an inside-out orientation.…”

“…Detailed kinetic studies have been carried out in rat heart mitochondria by Sluse et al (for reviews, see [4,5]), leading to an exceptional complexity in two aspects. While saturation of the carrier with external malate [6], or with internal oxoglutarate 151, could be described by a normal Michaelis-Menten curve, this was not possible for internal malate due to negative cooperativity and due to allosteric influences of other metabolites [5,7,81. In the case of external oxoglutarate a complex saturation curve was also observed [9], which was interpreted to reflect an oligomeric structure of the carrier protein [4 -61. In recent years, the oxoglutarate carrier has been purified from heart and liver mitochondria [lo, 111.…”

Anion Carriers of Mitochondrial Membranes

“…1) the internal oxoglutarate seems to be Michaelian for initial rate versus internal oxoglutarate concentration in intact mitochondria (apparent ~ I::l 1 mM) and for equilibrium binding for external oxoglutarate with inverted submitochondrial particles ~ I::l 800 I'M) while the internal-malate kinetic saturation curves reveal negative cooperatively modulated by the internal-aspartate concentrations (Fig. The exceptional behaviour of oxoglutarate translocator has been analysed and demonstrates that the translocator has an oligomeric structure (Sluse-Goffart et al, 1983) and that several conformational changes are involved during its activity cycle (Sluse-Goffart & Sluse, 1986;Duyckaerts et aL, 1984). The exceptional behaviour of oxoglutarate translocator has been analysed and demonstrates that the translocator has an oligomeric structure (Sluse-Goffart et al, 1983) and that several conformational changes are involved during its activity cycle (Sluse-Goffart & Sluse, 1986;Duyckaerts et aL, 1984).…”