Both the 25-kDa and 50-kDa domains in myosin subfragment 1 are close to the reactive thiols.
The thiol-specific photoactivatable reagent benzophenone-4-iodoacetamide can be incorporated into myosin subfragment 1 (S1), accompanied by an increase of Ca2+-ATPase and the loss of K+-ATPase activities, a characteristic property of S1 when reactive sulfhydryl 1 (SH-1) is modified. After trypsin cleavage, 25-kDa, 50-kDa, and 20-kDa fragments were found upon NaDodSO4/polyacrylamide gel electrophoresis of the unphotolyzed sample, whereas only the 50-kDa fragment and a 45-kDa fragment appeared in the photolyzed sample, indicating that the NH2-terminal 25-kDa fragment was crosslinked to the COOH-terminal 20-kDa fragment via SH-1. When photolysis was carried out in the presence of Mg2+ and ATP or Mg2+ and adenosine 5-[beta, gamma-imido]triphosphate (AdoPP[NH]P), a 70-kDa band, attributable to a crosslinked (50 kDa + 20 kDa) species, was also observed. This suggests that the conformational change induced by nucleotide binding reduces the distance between the 50-kDa region and the label on SH-1. Similar results were obtained when labeling and photolysis were carried out on trypsin-nicked S1, in which the 25-kDa, 50-kDa, and 20-kDa fragments are held together noncovalently. Further, when labeling with benzophenone-4-iodoacetamide was carried out in the presence of Mg-ATP, which increases the reactivity of another thiol, presumably SH-2, both 45-kDa and 70-kDa species were formed upon photolysis in the absence of ATP, suggesting that SH-2 is close to the 50-kDa region. More of the 70-kDa species was formed, at the expense of the 45-kDa species, when photolysis was carried out in the presence of Mg-ATP. Partial heat denaturation preferentially reduced the crosslinking between the reactive thiols and the 50-kDa region.
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001577:Benzophenones; D001665:Binding Sites; D000252:Calcium-Transporting ATPases / Q000378:metabolism; D027682:Cation Transport Proteins; D003432:Cross-Linking Reagents; D003545:Cysteine; D015879:Myosin Subfragments; D009218:Myosins; D010446:Peptide Fragments; D011487:Protein Conformation; D011817:Rabbits; D013329:Structure-Activity Relationship; D013439:Sulfhydryl Reagents
D001577:Benzophenones; D027682:Cation Transport Proteins; D003432:Cross-Linking Reagents; D015879:Myosin Subfragments; D010446:Peptide Fragments; D013439:Sulfhydryl Reagents; C049923:benzophenone-4-iodoacetamide; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; C022761:potassium transporting ATPase; D009218:Myosins; D000252:Calcium-Transporting ATPases; D003545:Cysteine