Labeling the adenine nucleotide binding domain of the sarcoplasmic reticulum Ca,Mg-ATPase with photoaffinity analogs of ATP.

Abstract The ATP analog, 3'-O-(4-benzoyl)benzoic adenosine triphosphate (BzATP) was an effective photoaffinity analog of ATP for labeling the sarcoplasmic reticulum Ca,Mg-ATPase. In contrast to 8-azido-ATP and arylazido-ATP, BzATP produced significant inhibition of ATPase activity. The incorporation of [alpha-32P]BzATP was restricted to the Ca,Mg-ATPase at 250 microM BzATP and the incorporation was antagonized by ATP. We conclude that the photodependent incorporation of BzATP is restricted to the active site because the inhibition of ATPase activity was stoichiometrically related to analog incorporation. Resolution of the tryptic fragments produced from photolabeled Ca,Mg-ATPase demonstrated that fragments A and B were labeled in a ratio of about 2:1. Both fragments must, therefore, contain portions of the active site.