Interaction of 4-Azido-2-Nitrophenyl Phosphate, a Pseudosubstrate, with the Sarcoplasmic Reticulum Ca-ATPase

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Abstract
In the dark, 4-azido-2-nitrophenyl phosphate (ANPP) is a phosphate analog which behaves like a simple energy-rich phosphate donor for the sarcoplasmic reticulum (SR) Ca-ATPase. Like p-nitrophenyl phosphate (pNPP), ANPP is hydrolyzed by the enzyme only in the presence of calcium and magnesium (K0.5 and Vmax are 0.3 mM and 60 nmol mg-1 min-1, respectively). After photoirradiation in the absence of magnesium, SR Ca-ATPase is specifically labeled by [32P]ANPP in the presence and in the absence of calcium. The presence of nucleotide in the medium provides some protection against photolabeling but less than phosphorylation by inorganic phosphate. The maximal stoichiometry of covalently bound ANPP extrapolates to 0.8 mol/mol of Ca-ATPase in the absence of magnesium. Autoradiography of a sodium dodecyl sulfate-polyacrylamide gel, after controlled trypsin digestion of the photolabeled protein, reveals that the radioactivity is incorporated in the B subfragment. Three radioactive polypeptides with approximate molecular masses of 55, 25, and 6 kDa are obtained depending on the digestion conditions. N-Terminal sequence analysis of the 50- and 25-kDa peptides reveals the same sequence beginning at Ala-506, whereas two different sequence beginning at Ala-506 and Phe-584 are observed in the 6-kDa peptides.

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