PHOTOAFFINITY-LABELING OF THE MITOCHONDRIAL OXOGLUTARATE CARRIER BY AZIDO-PHTHALONATE

The effect of azido-phthalonate, a photoreactive analogue of oxoglutar ate, on the transport of oxoglutarate was investigated in proteoliposo mes reconstituted with the purified oxoglutarate carrier. In the dark, azido-phthalonate inhibits the reconstituted oxoglutarate/oxoglutarat e exchange in a competitive manner with a K-i of 0.38 mM. Upon photoir radiation, the inhibition df the oxoglutarate exchange by azido-phthal onate is not removed by passing the proteoliposomes through a Sephadex column. The light-induced inhibition of the oxoglutarate/oxoglutarate exchange activity by azido-phthalonate is time- and concentration-dep endent. The kinetic analysis of transport inhibition by azido-phthalon ate reveals that one molecule of this substrate analogue bound to the functional carrier molecule is responsible for complete inhibition of the carrier function. Azido-[H-3]phthalonate binds to the oxoglutarate carrier covalently: Incubation of the proteoliposomes with oxoglutara te during photoirradiation in the presence of azido-phthalonate protec ts the carrier against inactivation and decreases the amount of radioa ctivity which is found to be associated with the carrier protein. It i s concluded that azido-phthalonate can be used for photoaffinity label ing of the mitochondrial oxoglutarate carrier at the substrate-binding site.