Synthesis and properties of azidonitrophenyl pyrophosphate, a photoaffinity probe of the nucleotide binding sites of mitochondrial F1-ATPase.
4-Azido-2-nitrophenyl pyrophosphate (azido-PPi) labeled with 32P in the alpha position was prepared and used to photolabel beef heart mitochondrial F1. Azido-PPi was hydrolyzed by yeast inorganic pyrophosphatase, but not by mitochondrial F1-ATPase. Incubation of F1 with [alpha-32P]azido-PPi in the dark under conditions of saturation resulted in the binding of the photoprobe to three sites, two of which exhibited a high affinity (Kd = 2 microM), the third one having a lower affinity (Kd = 300 microM). Mg2+ was required for binding. As with PPi [Issartel et al. (1987) J. Biol. Chem. 262, 13538-13544], the binding of 3 mol of azido-PPi/mol of F1 resulted in the release of one tightly bound nucleotide. ADP, AMP-PNP, and PPi competed with azido-PPi for binding to F1, but Pi and the phosphate analogue azidonitrophenyl phosphate did not. The binding of [32P]Pi to F1 was enhanced at low concentrations of azido-PPi, as it was in the presence of low concentrations of PPi. Sulfite, which is thought to bind to an anion-binding site on F1, inhibited competitively the binding of both ADP and azido-PPi, suggesting that the postulated anion-binding site of F1 is related to the exchangeable nucleotide-binding sites. Upon photoirradiation of F1 in the presence of [alpha-32P]azido-PPi, the photoprobe became covalently bound with concomitant inactivation of F1. The plots relating the inactivation of F1 to the covalent binding of the probe were rectilinear up to 50% inactivation.(ABSTRACT TRUNCATED AT 250 WORDS)
Michel
L
L
Département de Recherche Fondamentale, Centre d'Etudes Nucléaires, Grenoble, France.
Garin
J
J
Issartel
J P
JP
Vignais
P V
PV
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001386:Azides / Q000138:chemical synthesis*; D001665:Binding Sites; D001667:Binding, Competitive; D002417:Cattle; D004789:Enzyme Activation; D008929:Mitochondria, Heart / Q000201:enzymology; D009711:Nucleotides / Q000378:metabolism; D009943:Organophosphorus Compounds / Q000138:chemical synthesis*; D010761:Phosphorus Radioisotopes; D010777:Photochemistry; D006180:Proton-Translocating ATPases / Q000378:metabolism; D011755:Pyrophosphatases / Q000378:metabolism; D013447:Sulfites / Q000378:metabolism
D001386:Azides; D009711:Nucleotides; D009943:Organophosphorus Compounds; D010761:Phosphorus Radioisotopes; D013447:Sulfites; C063118:4-azido-2-nitrophenyl pyrophosphate; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D011755:Pyrophosphatases; D006180:Proton-Translocating ATPases