Photolabeling of the 6 and 10 S conformations of gizzard myosin with 3'(2')-O-(4-Benzoyl)benzoyl-ATP. Proline 324 is near the active site.

D G Cole,R G Yount

doi:10.1016/s0021-9258(18)45739-0

Abstract

3'(2')-O-(4-Benzoyl)benzoyl-ATP (Bz2ATP) was used as a photoaffinity label of the ATP binding site of unphosphorylated chicken gizzard myosin. Specific photolabeling of the active site of 6 S myosin was assured by forming a stable myosin.Co(II)Bz2ADP.orthovanadate complex (termed trapping) prior to irradiation. Co2+ was used in place of Mg2+ to prevent the known photoreaction of vanadate with myosin which destabilizes the trapped complex. [3H] Bz2ADP.Pi was also stably trapped on gizzard myosin by forming the 10 S folded conformation of the protein in the presence of [3H]Bz2ATP and Mg2+. Irradiation of 6 S myosin containing orthovanadate trapped [3H] Bz2ADP or 10 S trapped [3H]Bz2ADP.Pi gave 32 and 30% covalent incorporation, respectively. The 50-kDa and precursor 68-kDa tryptic peptides of the subfragment-1 heavy chain derived from both forms of myosin were found to contain essentially all of the covalently attached [3H]Bz2ADP. Parallel experiments with untrapped [3H]Bz2ADP showed extensive nonspecific labeling of all of the major tryptic peptides and the light chains. Eight labeled peptides, isolated from 6 and 10 S photolabeled myosin, contained the sequence G319-H-V-P-I-X-A-Q326, where X corresponds to labeled proline 324. [14C]Bz2ADP was previously shown to label serine 324 in skeletal subfragment-1 (Mahmood, R., Elzinga, M., and Yount, R. G. (1989) Biochemistry 28, 3989-3995), which corresponds to alanine 325 in the gizzard sequence. Thus, this region of the 50-kDa tryptic fragment, near the nucleotide binding site, in both skeletal and smooth muscle myosins, must fold in essentially the same manner.

Highlights

Was used as a photoaffinity label of the ATP binding site of unphosphorylated chicken gizzard myosin
Photolabeling is shown to be highly specific in both cases and to label a specific proline in the 50-kDa tryptic fragment in a position analogous to that observed previously with Bz*ADP photolabeling of skeletal myosin (Mahmood et al, 1989). These results suggest that the part of the 50-kDa fragment which is near the ATP binding site folds in an identical manner in both smooth and skeletal myosins
The purpose of this work was to probe for possible differences in the active site region of 6 and 10 S gizzard myosin with the photoaffinity label, Bz2ATP

Summary

Introduction

Was used as a photoaffinity label of the ATP binding site of unphosphorylated chicken gizzard myosin. Bz2ADP*Pi was stably trapped on gizzard myosin by forming the 10 S folded conformation of the protein in the presence of [3H]Bz2ATP and M&+. The BO-kDa and precursor 6%kDa tryptic peptides of the subfragmerit- 1 heavy chain derived from both forms of myosin were found to contain essentially all of the covalently attached [3H]Bz2ADP. Eight labeled peptides, isolated from 6 and 10 S photolabeled myosin, contained the sequence. G. (1989) Biochemistry 28, 3989-3995), which corresponds to alanine 325 in the gizzard sequence. This region of the BO-kDa tryptic fragment, near the nucleotide binding site, in both skeletal and smooth muscle myosins, must fold in essentially the same manner

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Conclusion