Affinity of nucleotides for the active site of detergent-solubilized sarcoplasmic reticulum CaATPase

A dye displacement method was developed for determination of the affinities of compounds toward the active site of the detergent-solubilized sarcoplasmic reticulum CaATPase. Titration of the enzyme with 2',3'-O-(2,4,6-trinitrophenyl)-ADP resulted in a large absorbance difference in the visible spectrum. Subsequent addition of compounds which bind to the active site causes displacement of the dye; resulting absorbance changes were treated to gain dissociation constants for added ligands. The active site affinities of ATP, and nonhydrolyzable nucleotide analogues were determined. Both occupancy of the high affinity calcium site and added divalent cations have large influences on nucleotide affinity.