Kinetic and energetic characterization of solute flux through the reconstituted aspartate/glutamate carrier from beef heart mitochondria after modification with mercurials

摘要

The functional switch from specific, coupled antiport to unspecific unidirectional transport (efflux) of the reconstituted aspartate/glutamate carrier from mitochondria after chemical modification with mersalylic acid was investigated in kinetic and energetic terms. The rate of mercurial-induced efflux was determined for a number of solutes which differ from the physiological substrate aspartate in structure, size and charge, namely oxoglutarate, sulfate, glucose, lysine and arginine. These values were compared to the rates of efflux as well as antiport of aspartate. Measurement of the temperature dependence of all rates led to evaluation of the activation energy of the different substrates. The activation energy was similar for all substrates and for both transport modes, whereas the efflux rates could be ordered in the following sequence: anions > uncharged solutes > cations. When extrapolating to Vmax conditions, the resulting turnover numbers for uniport substrates become similar and exceed the turnover numbers for aspartate and glutamate antiport. Trans-inhibition of efflux was only observed in the case of externally added aspartate or glutamate and only for internal anionic substrates (at the cis side), thus indicating that after efflux induction the specificity of the external binding site is fully and that of the internal site is partially retained. The consequence of these results for understanding the transport function of the aspartate/glutamate carrier in the slippage mode (uniport) is discussed in energetic and kinetic terms.