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The aim of the study was to investigate the effect of in vitro anoxia/reoxygenation on the oxidative phosphorylation of isolated lung mitochondria. Mitochondria were isolated after harvesting from fresh pig lungs flushed with Euro-Collins solution. Mitochondrial respiratory parameters were determined in isolated mitochondria before anoxia (control), after 5-45 min anoxia followed by 5 min reoxygenation, and after 25 or 40 min of in vitro incubation in order to follow the in vitro aging of mitochondria during respiratory assays. Respiratory parameters measured after anoxia/reoxygenation did not show any oxidative phosphorylation dysfunction, indicating a high resistance of pulmonary mitochondria to in vitro anoxia/reoxygenation (up to 45 min anoxia). These results indicate that mitochondria are not directly responsible of their oxidative phosphorylation damage observed after in vivo ischemia (K. Willet et al., Transplantation 69 (2000) 582) but are a target of others cellular injuries leading to mitochondrial dysfunction in vivo.

Interaction of glutamate and aspartate with the oxoglutarate carrier was investigated in rat heart mitochondria or inverted submitochondrial particles. With mitochondria, glutamate and aspartate had no effect on the initial rate of oxoglutarate or malate uptake. With inverted submitochondrial vesicles, binding experiments indicated that aspartate bound to the oxoglutarate carrier on its matricial face and increased the affinity of the substrate binding site for malate but did not change the affinity for oxoglutarate. Glutamate had no effect on both substrate bindings. The dissociation constants of the binary substrate-carrier complexes on the matricial side were determined (1.28 +/- 0.15 mM for oxoglutarate and 2.22 +/- 0.26 mM for malate). These values, compared with those obtained previously on the cytosolic side of intact mitochondria, confirmed the asymmetry of the carrier in the native membrane (higher affinities on the cytosolic face). It is concluded that (1) aspartate and glutamate are not cytosolic effectors of the oxoglutarate carrier, (2) matricial aspartate is a positive effector of the binding of malate on the matricial side of the oxoglutarate carrier, and (3) such a characteristic may play a role in the regulation of the oxoglutarate carrier. Thus, it may be emphasized that (1) this observation is the first clear evidence of a well-defined 'sophisticated regulation' (allosteric) of a mitochondrial metabolite carrier, and (2) this regulation of the oxoglutarate carrier may have important consequences on the efficiency of reducing equivalent import in the matrix space by the malate-aspartate shuttle.

The homologous exchange of external [14C] aspartate/internal aspartate catalyzed by the aspartate/glutamate carrier of rat heart mitochondria was investigated using aspartate-loaded, glutamate-depleted mitochondria. An inhibitor-stop technique was developed for kinetic studies by applying pyridoxal phosphate. Direct initial rate determinations from the linear phase of [14C] aspartate uptake were insufficiently accurate at high external and/or low internal substrate concentrations. Therefore, the full time-course of [14C] aspartate uptake until reaching isotope equilibrium was fitted by a single exponential function and was used to calculate reliable initial steady-state rates. This method was applied in bisubstrate analyses of the antiport reaction for different external and internal aspartate concentrations. The kinetic patterns obtained in double reciprocal plots showed straight lines converging on the abscissa. This result is consistent with a sequential antiport mechanism. It implies the existence of a catalytic ternary complex that is formed by the translocator and substrate molecules bound from both sides of the membrane. The Km values for aspartate were clearly different for the external and the internal sides of the membrane, 216 +/- 23 microM and 2.4 +/- 0.5 mM, respectively. These values indicated a definite transmembrane asymmetry of the carrier. The same asymmetry became evident when investigating the isolated protein from bovine heart mitochondria after reconstitution into liposomes. In this case the Km values for external and internal aspartate were determined to be 123 +/- 11 microM and 2.8 +/- 0.6 mM, respectively. This comparison demonstrates a right-side out orientation of the carrier after insertion into liposomal membranes. The sequential transport mechanism of the aspartate/glutamate carrier, elucidated both in proteoliposomes and in mitochondria, also seems to be a common characteristic of other mitochondrial antiport carriers.