Kinetics and regulation of the glutamate-aspartate translocator in rat liver mitochondria.

The kinetics and mechanism of the electrogenic exchange of external glutamate with intramitochondrial aspartate have been investigated using aspartateloaded rat liver mitochondria in the absence of metabolism. Apparent kinetic constants were calculated from measured decreases of the mitochondrial matrix aspartate content after glutamate addition by a computer curve fitting procedure and by graphical analysis. An increase of external glutamate concentration caused an increase of V’,,, but only small changes of the K’, for matrix aspartate. The decrease of the K’,/V’,,, ratio with increasing glutamate concentratior. rules out a ping-pong mechanism for the translocator, and indicates a ternary complex between the carrier, glutamate and aspartate. The dependencies of aspartate efflux kinetics on the mitochondrial membrane potential (A*) and ApH were also investigated in aspartate-loaded mitochondria. The V&,, of aspartate efflux was not affected by changes of the external pH over the range from 6.5 to 7.6 or by changes of the internal pH over the range of 6.8 to 9.0. However, the V’,,, of aspartate efflux was linearly dependent on A\k over the range from 70 to 160 mV. Under all conditions investigated, including the de-energized state, the K’, of the glutamate-aspartate translocator for matrix aspartate remained approximately constant. These data indicate (a) binding of uncharged glutamate to the carrier, and (b) that the translocation of a negatively charged carrier l aspartate complex across the mitochondrial membrane is the rate-limiting step of the reaction sequence. Glutamate-independent kinetic constants were calculated from the apparent kinetic constants obtained from the computer and graphical fitting procedures. Average values were 3.9 nmol/mg for the matrix aspartate K,,,, 5.8 InM for the external glutamate K,,,, and 21.2 nmol/mg/min for the Vmax at 10°C. Using these constants, together with a Ki for external aspartate of 4 InM, and a temperature coefficient of 15.1 kcal/mol, the aspartate translocator kinetics were described in terms of a rate equation. Values of mitochondrial aspartate efflux in isolated hepatocytes calculated using this rate equation were in approximate agreement with measured rates. It is concluded that no significant mi-