Photoaffinity labeling of the mitochondrial oxoglutarate carrier by azido-phthalonate.
The effect of azido-phthalonate, a photoreactive analogue of oxoglutarate, on the transport of oxoglutarate was investigated in proteoliposomes reconstituted with the purified oxoglutarate carrier. In the dark, azido-phthalonate inhibits the reconstituted oxoglutarate/oxoglutarate exchange in a competitive manner with a Ki of 0.38 mM. Upon photoirradiation, the inhibition of the oxoglutarate exchange by azido-phthalonate 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-dependent. The kinetic analysis of transport inhibition by azido-phthalonate reveals that one molecule of this substrate analogue bound to the functional carrier molecule is responsible for complete inhibition of the carrier function. Azido-[3H]phthalonate binds to the oxoglutarate carrier covalently. Incubation of the proteoliposomes with oxoglutarate during photoirradiation in the presence of azido-phthalonate protects the carrier against inactivation and decreases the amount of radioactivity which is found to be associated with the carrier protein. It is concluded that azido-phthalonate can be used for photoaffinity labeling of the mitochondrial oxoglutarate carrier at the substrate-binding site.
Stipani
I
I
Department of Pharmaco-Biology, University of Bari, Italy.
Natuzzi
D
D
Daddabbo
L
L
Ritieni
A
A
Randazzo
G
G
Palmieri
F
F
D000345:Affinity Labels; D000818:Animals; D002352:Carrier Proteins / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel; D007656:Ketoglutaric Acids / Q000378:metabolism*; D007700:Kinetics; D008081:Liposomes; D026901:Membrane Transport Proteins; D008929:Mitochondria, Heart / Q000378:metabolism*; D008970:Molecular Weight; D010795:Phthalic Acids / Q000138:chemical synthesis / Q000494:pharmacology*; D011510:Proteolipids / Q000378:metabolism
D000345:Affinity Labels; D002352:Carrier Proteins; D007656:Ketoglutaric Acids; D008081:Liposomes; D026901:Membrane Transport Proteins; D010795:Phthalic Acids; D011510:Proteolipids; C092806:azidophthalonate; C021723:oxoglutarate translocator; C020485:proteoliposomes