title: Studies of the nucleotide-binding sites on the mitochondrial F1-ATPase through the use of a photoactivable derivative of adenylyl imidodiphosphate.
abstract: (1)N-4-Azido-2-nitrophenyl-gamma-[3H]aminobutyryl-AdoPP[NH] P(NAP4-AdoPP[NH]P) a photoactivable derivative of 5-adenylyl imidodiphosphate (AdoPP[NH]P), was synthesized. (2) Binding of [3H]NAP4-AdoPP[NH]P to soluble ATPase from beef heart mitochondria (F1) was studied in the absence of photoirradiation, and compared to that of [3H]AdoPP[NH]P. The photoactivable derivative of AdoPP[NH]P was found to bind to F1 with high affinity, like AdoPP[NH]P. Once [3H]NAP4-AdoPP[NH]P had bound to F1 in the dark, it could be released by AdoPP[NH]P, ADP and ATP, but not at all by NAP4 or AMP. Furthermore, preincubation of F1 with unlabeled AdoPP[NH]P, ADP, or ATP prevented the covalent labeling of the enzyme by [3H]NAP4-AdoPP[NH]P upon photoirradiation. (3) Photoirradiation of F1 by [3H]NAP4-AdoPP[NH]P resulted in covalent photolabeling and concomitant inactivation of the enzyme. Full inactivation corresponded to the binding of about 2 mol [3H]NAP4-AdoPP[NH]P/mol F1. Photolabeling by NAP4-AdoPP[NH]P was much more efficient in the presence than in the absence of MgCl2. (4) Bound [3H]NAP4-AdoPP[NH]P was localized on the alpha- and beta- subunits of F1. At low concentrations (less than 10 microM), bound [3H]NAP4-AdoPP[NH]P was predominantly localized on the alpha-subunit; at concentrations equal to, or greater than 75 microM, both alpha- and beta-subunits were equally labeled. (5) The extent of inactivation was independent of the nature of the photolabeled subunit (alpha or beta), suggesting that each of the two subunits, alpha and beta, is required for the activity of F1. (6) The covalently photolabeled F1 was able to form a complex with aurovertin, as does native F1. The ADP-induced fluorescence enhancement was more severely inhibited than the fluorescence quenching caused by ATP. The precentage of inactivation of F1 was virtually the same as the percentage of inhibition of the ATP-induced fluorescence quenching, suggestion that fluorescence quenching is related to the binding of ATP to the catalytic site of F1.
mesh_terms: D000244:Adenosine Diphosphate / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000494:pharmacology; D000266:Adenylyl Imidodiphosphate / Q000031:analogs & derivatives* / Q000494:pharmacology; D000818:Animals; D001386:Azides; D001665:Binding Sites; D002417:Cattle; D004492:Edetic Acid / Q000494:pharmacology; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D015636:Magnesium Chloride; D008929:Mitochondria, Heart / Q000201:enzymology*; D011485:Protein Binding; D006180:Proton-Translocating ATPases / Q000378:metabolism*
chemical_list: D001386:Azides; D015636:Magnesium Chloride; D000266:Adenylyl Imidodiphosphate; D000244:Adenosine Diphosphate; C036313:N-4-azido 2-nitrophenyl gamma-aminobutyryl-5-adenylyl imidodiphosphate; D000255:Adenosine Triphosphate; D004492:Edetic Acid; D006180:Proton-Translocating ATPases; D008274:Magnesium