Photomodification of Mitochondrial Proteins by Azido Fatty Acids and Its Effect on Mitochondrial Energetics

Abstract

Azido derivatives of long-chain fatty acids, 12-(4-azido-2-nitrophenylamino)dodecanoic acid (N3-NpNH-Lau) and 16-(4-azido-2-nitrophenyla~ino)hexadecanoic acid (N,-NpNH-Pam), were used to study the mechanism of the protonophoric function of long-chain fatty acids in mitochondrial membranes. N,-NpNH-Lau was found to increase resting-state respiration and decrease the membrane potential in a dosedependent way in a manner similar to that of the natural fatty acid, myristate. Both effects of N,-NpNHLau as well as of the myristate were reversed or prevented by the inhibitor of the mitochondrial ADP/ATP carrier (AAC), carboxyatractyloside. This protective effect of carboxyatractyloside was well expressed in rat heart mitochondria and less so in mitochondria within digitonin-permeabilized Ehrlich ascites tumour cells. Photomodification of Ehrlich ascites tumour mitochondria by ultraviolet irradiation in the presence of N,-NpNH-Lau made them more resistant to the uncoupling effect of myristate, and photomodification of rat heart mitochondria resulted in a strong inhibition of AAC which could not be reversed by serum albumin.Photolabelling of rat heart mitochondria with tritiated N,-NpNH-Pam revealed around 10 labelled bands on SDS/polyacrylamide gel electrophoresis. Based on immunodetection with a specific antibody, one of them, corresponding to 30 kDa, was identified as AAC. Specific interaction of AAC with azido fatty acids was confirmed by a high radiolabelling of this band.The role of fatty acids in fine control of the efficiency of oxidative phosphorylation is discussed.Keywords: fatty acid ; ADP/ATP carrier; protonophoric activity ; uncoupling ; mitochondria.Long-chain fatty acids (in their activated form) are readily oxidized by mitochondria of most mammalian tissues, feeding electrons to the respiratory chain and thus contributing to energy production in the form of ATP. However, fatty acids also interact with mitochondrial membranes and interfere with the energycoupling system as weak uncouplers. Thus, they may play a dual role in cell energetics, being metabolites as well as regulators of oxidative phosphorylation efficiency. The uncoupling effect of fatty acids has been well known for four decades (for review, see [I]). However, the mechanism by which fatty acids decrease the efficiency of energy transformation in mitochondria is not fully understood. A direct protonophoric mechanism has been proposed [2-51, but objections have been raised based on a very slow flip-flop rate of the fatty acid anion [2,

Adenine nucleotide translocase family: four isoforms for apoptosis modulation in cancer

“…This supports the hypothesis that ANT participates to a first line of defense against ROS in response to fatty acids, superoxide and lipid peroxidation products under conditions of high oxidative damage (Figure 1) (Scho¨nfeld et al, 1996;Khailova et al, 2006;Azzu et al, 2008;Jastroch et al, 2010;Toime and Brand, 2010). This ANT , 2003).…”

Reconstituted Plant Uncoupling Mitochondrial Protein Allows for Proton Translocation via Fatty Acid Cycling Mechanism

“…4 and 5) (2) and induction of H ϩ transport exclusively with FAs having the ability to flip-flop across the lipid bilayer in a protonated form, suggest that PUMP conducts FA anions and, consequently, allows for FA cycling, which leads to uncoupling (6). PUMP is the third example of an integral membrane protein possessing this ability, besides the mammalian uncoupling protein UcP (6) and the ADP/ATP carrier (19,20).…”

Natural and Azido Fatty Acids Inhibit Phosphate Transport and Activate Fatty Acid Anion Uniport Mediated by the Mitochondrial Phosphate Carrier

“…Consequently, FAs should be considered as important regulators of oxidative phosphorylation (29), since they affect PIC (Ref. 20 and this work) and AAC (18,19,21,22), besides the other mitochondrial carriers (30) and uncoupling proteins (19,(23)(24)(25)(26)(27).…”

“…Also, the 30-kDa band in these fractions retained the 3 H-AzHA label. It was identified as AAC by Western blots (22). The "upper" bands were found only in minute amounts in the intermediate NaCl fractions of the BS column.…”

“…7). These two most likely correspond to AAC (22) and PIC, respectively. Our further steps led to the verification of this assumption.…”