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

Abstract

4-Azido-2-nitrophenyl phosphate (ANPP) was synthesized and characterized. ANPP, unlabeled or labeled by 32P, was used as a photoreactive analog 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 Fi-ATPase by ANPP in the dark; the Ki value was 60 .mu.M. Upon photoirradiaiton with visible light, [32P]ANPP bound covalently to F1-ATPase and inactivated the enzyme. Part of the added ANPP was photolyzed with release of Pi. By extrapolation, it could be calculated that complete inactivation of F1-ATPase was accompanied by incorporation of 32P radioactivity corresponding to 1 mol of [32P]ANPP/mol of F1-ATPase. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of [32P]-ANPP-labeled F1-ATPase revealed only one radioactive peptide with a MW of 50,000. This peptide was characterized as the .beta. subunit of F1-ATPase by specific labeling with [14C]dicyclohexylcarbodiimide. Photoirradiation of inside-out submitochondrial particles with [32P]ANPP results in the labeling of 2 peptides with a MW of 50,000 and 30,000-32,000; both labelings were significantly decreased by incubation of the particles with Pi prior to photoirradiation. The MW 50,000 peptide is probably the .beta. subunit of F1-ATPase; the other peptide might be the Pi carrier protein.