Photosensitized direct cross-linking of fluorescent analogs of ATP to the adenine recognition domain in myosin ATPase.

To obtain information about the adenine recognition site in myosin ATPase, ribosemodified fluorescent analogs of ATP, 3'-O-anthraniloyl and 3'-O-(N-methylanthraniloyl) derivatives, were directly cross-linked to myosin subfragment-1 (S-1) ATPase by irradiation with visible light in the presence of FMN as a photosensitizer. The cross-linking of the fluorescent nucleotides was inhibited by addition of ATP or ADP. Tryptic digestion of the cross-linked S-1 revealed that fluorescence of the analog was associated predominantly with the 50K fragment and its precursor, the 75K one, and slightly with the 20K fragment. However, fluorescence was scarcely associated with the 26K fragment. The results were confirmed by cross-linking experiments using trypsin-split S-1, which mainly consists of the 50K, 26K, and 20K fragments. These findings suggest that the adenine recognition site of the myosin ATPase is located predominantly on the 50K domain.