PHOTOMODIFICATION OF MITOCHONDRIAL PROTEINS BY AZIDO FATTY-ACIDS AND ITS EFFECT ON MITOCHONDRIAL ENERGETICS - FURTHER EVIDENCE FOR THE ROLEOF THE ADP ATP CARRIER IN FATTY-ACID-MEDIATED UNCOUPLING/

Azido derivatives of long-chain fatty acids, 12-(4-azido-2-nitrophenyl amino)dodecanoic acid (N-3-NpNH-Lau) and 16-(4-azido-2-nitrophenylamin o)hexadecanoic acid (N-3-NpNH-Pam), were used to study the mechanism o f the protonophoric function of long-chain fatty acids in mitochondria l membranes. N-3-NpNH-Lau was found to increase resting-state respirat ion and decrease the membrane potential in a dose-dependent way in a m anner similar to that of the natural fatty acid, myristate. Both effec ts of N-3-NpNH-Lau as well as of the myristate were reversed or preven ted by the inhibitor of the mitochondrial ADP/ATP carrier (AAC), carbo xyatractyloside. This protective effect of carboxyatractyloside was we ll expressed in rat heart mitochondria and less so in mitochondria wit hin digitonin-permeabilized Ehrlich ascites tumour cells. Photomodific ation of Ehrlich ascites tumour mitochondria by ultraviolet irradiatio n in the presence of N-3-NpNH-Lau made them more resistant to the unco upling effect of myristate, and photomodification of rat heart mitocho ndria resulted in a strong inhibition of AAC which could not be revers ed by serum albumin. Photolabelling of rat heart mitochondria with tri tiated N-3-NpNH-Pam revealed around 10 labelled bands on SDS/polyacryl amide gel electrophoresis. Based on immunodetection with a specific an tibody, one of them, corresponding to 30 kDa, was identified as AAC. S pecific interaction of AAC with azido fatty acids was confirmed by a h igh radiolabelling of this band. The role of fatty acids in fine contr ol of the efficiency of oxidative phosphorylation is discussed.