4-Azido-2-nitrophenyl phosphate, a new photoaffinity derivative of inorganic phosphate. Study of its interaction with the inorganic phosphate binding site of beef heart mitochondrial adenosine triphosphatase.
4-Azido-2-nitrophenyl phosphate (ANPP) was synthesized and characterized. ANPP, unlabeled or labeled by 32P, was used as a photoreactive analogue of Pi to study the Pi binding site(s) in isolated F1-ATPase and inside-out particles from beef heart mitochondria. In the dark, the phosphate bond of ANPP was cleaved by alkaline phosphatase but not by mitochondrial F1-ATPase. ANPP bound reversibly to the phosphate site of F1-ATPase as shown by competitive inhibition of binding of Pi to F1-ATPase by ANPP in the dark; the Ki value was 60 microM. Upon photoirradiation with visible light, [32P]ANPP bound covalently to F1-ATPase and inactivated the enzyme. Part of the added ANPP was, however, photolyzed with release of Pi. By extrapolation, it could be calculated that complete inactivatin of F1-ATPase was accompanied by incorporation of 32P radioactivity corresponding to 1 mol of [32P]ANPP per mol of F1-ATPase. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of [32P]-ANPP-labeled F1-ATPase revealed only one radioactive peptide with a Mr of 50000. This peptide was characterized as the beta subunit of F1-ATPase by specific labeling with [14C]dicyclohexylcarbodiimide [Pougeois, R., Satre, M., & Vignais, P. V. (1979) Biochemistry 18, 1408-1413]. Photoirradiation of inside-out submitochondrial particles with [32P]ANPP resulted in the labeling of two peptides with a Mr of 50000 and 30000-32000; both labelings were significantly decreased by incubation of the particles with Pi prior to photoirradiation. The Mr 50000 peptide is most probably the beta subunit of F1-ATPase; the other peptide might be the Pi carrier protein.
D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000345:Affinity Labels; D000818:Animals; D001386:Azides / Q000378:metabolism* / Q000528:radiation effects; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel; D008027:Light; D008929:Mitochondria, Heart / Q000201:enzymology*; D010710:Phosphates / Q000378:metabolism*; D010761:Phosphorus Radioisotopes; D006180:Proton-Translocating ATPases; D012967:Sodium Dodecyl Sulfate
D000345:Affinity Labels; D001386:Azides; D010710:Phosphates; D010761:Phosphorus Radioisotopes; D012967:Sodium Dodecyl Sulfate; C027150:4-azido-2-nitrophenyl phosphate; D000251:Adenosine Triphosphatases; D006180:Proton-Translocating ATPases