Photoaffinity labeling of mitochondrial adenosine triphosphatase by an azido derivative of the natural adenosine triphosphate inhibitor.
The natural mitochondrial ATPase inhibitor (IF1) was modified with a radioactivity labeled heterobifunctional and photosensitive reagent, methyl 4-azido(14C)benzimidate ((14C)MABI). Titration experiments of IF1 by (14C)MABI and tryptic maps of (14C)MABI-IF1 indicated that specific lysine residues in IF1 are preferentially labeled by (14C)MABI. Under appropriate conditions of labeling (1 to 2 lysine residues modified per IF1), MABI-IF1 exhibited the same inhibitory potency as native IF1 on the hydrolytic activity of the coupling factor 1 of mitochondrial ATPase (F1). The same conditions were required for inhibition of F1 by MABI-IF1 and IF1 (slightly acidic pH and presence of ATP and MgCl2). In photolabeling experiments, (14C)MABI-IF1 was used to investigate the localization of IF1 binding sites on F1. Upon photoirradiation, MABI-IF1 bound selectively to the beta subunit of soluble or membrane-bound F1. Adenylyl imidodiphosphate and quercetin, two compounds which partially mimic the inhibitory effect of IF1 on ATPase activity of F1, markedly prevented the binding of (14C)MABI-IF1 to F1; on the other hand, aurovertin, a specific ligand of the beta subunit of F1, did not affect the interaction between (14C)MABI-IF1 and F1. In the absence of light, (14C)MABI-IF1 was used as a reversible radiolabeled ligand with respect to membrane bound F1 to investigate F1-IF1 interactions to inside-out submitochondrial particles as a function of the energy state of the particles. Oxidation of NADH by submitochondrial particles resulted in a decrease of bound (14C)MABI-IF1; the effect was counteracted by antimycin. The data suggested that added (14C)MABI-IF1 is capable of exchanging with IF1 bound to F1 in submitochondrial particles and that the rate and extent of (14C)MABI-IF1 release are triggered by the proton-motive force developed by the particles.
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000345:Affinity Labels; D000818:Animals; D002417:Cattle; D003432:Cross-Linking Reagents; D007700:Kinetics; D008929:Mitochondria, Heart / Q000201:enzymology*; D008970:Molecular Weight; D010446:Peptide Fragments / Q000032:analysis; D010777:Photochemistry; D011506:Proteins / Q000494:pharmacology; D013056:Spectrophotometry, Ultraviolet
D000345:Affinity Labels; D003432:Cross-Linking Reagents; D010446:Peptide Fragments; D011506:Proteins; C030007:methyl-4-azidobenzimidate ATPase inhibitor IF(1); D000251:Adenosine Triphosphatases