Photomodification of mitochondrial proteins by azido fatty acids and its effect on mitochondrial energetics. Further evidence for the role of the ADP/ATP carrier in fatty-acid-mediated uncoupling.
Azido derivatives of long-chain fatty acids, 12-(4-azido-2-nitrophenylamino)dodecanoic acid (N3-NpNH-Lau) and 16-(4-azido-2-nitrophenylamino)hexadecanoic acid (N3-NpNH-Pam), were used to study the mechanism of the protonophoric function of long-chain fatty acids in mitochondrial membranes. N3-NpNH-Lau was found to increase resting-state respiration and decrease the membrane potential in a dose-dependent way in a manner similar to that of the natural fatty acid, myristate. Both effects of N3-NpNH-Lau 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 N3-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 N3-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.
Schönfeld
P
P
Institute of Biochemistry, Otto-von-Guericke University, Magdeburg, Germany.
Jezek
P
P
Belyaeva
E A
EA
Borecký
J
J
Slyshenkov
V S
VS
Wieckowski
M R
MR
Wojtczak
L
L
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000345:Affinity Labels; D000818:Animals; D001386:Azides; D015153:Blotting, Western; D002286:Carcinoma, Ehrlich Tumor; D005227:Fatty Acids / Q000378:metabolism* / Q000494:pharmacology*; D005260:Female; D008564:Membrane Potentials / Q000187:drug effects; D008565:Membrane Proteins / Q000378:metabolism*; D008928:Mitochondria / Q000378:metabolism*; D008929:Mitochondria, Heart / Q000378:metabolism*; D000226:Mitochondrial ADP, ATP Translocases / Q000378:metabolism*; D010101:Oxygen Consumption / Q000187:drug effects; D010777:Photochemistry; D051381:Rats; D014407:Tumor Cells, Cultured; D014466:Ultraviolet Rays
D000345:Affinity Labels; D001386:Azides; D005227:Fatty Acids; D008565:Membrane Proteins; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000226:Mitochondrial ADP, ATP Translocases