Direct photoaffinity labeling of gizzard myosin with vanadate-trapped adenosine diphosphate

The active-site topology of smooth muscle myosin has been investigated by direct photoaffinity-labeling studies with 3H]ADP. Addition of vanadate (Vi) and Co2+ enabled 3H]ADP to be stably trapped at the active site (t1/2 greater than 5 days at 0 degrees C). The extraordinary stability of the myosin.Co2+.3H]ADP.Vi complex allowed it to be purified free of excess 3H]ADP before irradiation began and ensured that only active-site residues became labeled. Following UV irradiation, approximately 10% of the trapped 3H]ADP became covalently attached at the active site. All of the 3H]ADP incorporated into the 200-kDa heavy chain, confirming earlier results using untrapped alpha-32P]ATP Maruta, H., & Korn, E. (1981) J. Biol. Chem. 256, 499-502]. After extensive trypsin digestion of labeled subfragment 1, HPLC separation methods combined with alkaline phosphatase treatment allowed two labeled peptides to be isolated. Sequence analysis of both labeled peptides indicated that Glu-185 was the labeled residue. Since Glu-185 has been previously identified as a residue at the active site of smooth myosin using 3H]UDP as a photolabel Garabedian, T. E., & Yount, R. G. (1990) J. Biol. Chem. 265, 22547-22553], these results provide further evidence that Glu-185, located immediately adjacent to the glycine-rich loop, is located in the purine binding pocket of the active site of smooth muscle myosin.