Photoaffinity labelling of gizzard myosin with 3'-O-(4-benzoyl)-benzoic-adenosine 5'-triphosphate

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Abstract
The reaction of a photoaffinity analog, 3′‐O‐(4‐benzoyl)‐benzoic‐adenosine 5′‐triphosphate (Bz2ATP) with gizzard myosin is described. The incorporation of Bz2ATP into myosin is both specific and stoichiometric. About 2.2 mol Bz2ATP are incorporated/mol myosin resulting in the significant loss of EDTA(K+) ATPase activity. The Mg2+ and actin‐activated ATPase activities are slightly inhibited. Addition of ATP (millimolar) during the photolysis reaction significantly inhibits incorporation of Bz2ATP into myosin. Our data show that the label is mainly incorporated into the heavy chain of myosin with some label in the 20‐kDa light chain. Limited proteolysis of radioactively labeled myosin subfragment 1 with trypsin reveals the presence of radioactivity mainly in the 50‐kDa fragment and some in the 29‐kDa and 25‐kDa fragments. However, our data on the ATP‐sensitive incorporation of Bz2ATP into the tryptic fragments suggest that the 50‐kDa peptide, not the 29‐kDa peptide, may be located at or around the active site.