Photoaffinity labelling of smooth-muscle myosin by methylanthraniloyl-8-azido-ATP.
Methylanthraniloyl-8-azido-ATP (Mant-8-N3-ATP), which binds to the 20 kDa C-terminal tryptic fragment of skeletal-muscle myosin subfragment-1 [Maruta, Miyanishi and Matsuda (1989) Eur. J. Biochem. 184, 213-221], was synthesized and used as a probe of the conformational change of smooth-muscle myosin. Mant-8-N3-ATP, like ATP, induced the formation of the 10 S conformation at low ionic strength. In the presence of vanadate, smooth-muscle myosin formed a stable complex with Mant-8-N3-ADP, and this complex showed the 10 S-->6 S transition of myosin. ATP-binding sites for 6 S (extended state) and 10 S (folded state) myosin were studied by photolabelling of myosin with Mant-8-N3-ADP. For both 6 S and 10 S myosin, Mant-8-N3-ATP was incorporated into the 29 kDa N-terminal tryptic fragment of myosin heavy chain. This is unlike the labelling of skeletal-muscle myosin, in which the 20 kDa C-terminal fragment is labelled. The labelling of 29 kDa fragment was diminished significantly by addition of ATP. These results suggest that the conformation of the ATP-binding site of smooth-muscle myosin is different from that of skeletal-muscle myosin. To examine further the possible differences in the labelling site between 6 S and 10 S myosin, the affinity-labelled 29 kDa fragment was subjected to complete proteolysis by lysylendo-peptidase. The fluorescent-labelled-peptide map suggested that the Mant-8-N3-ADP-binding sites for 6 S and 10 S myosin were identical.
Department of Physiology and Biophysics, School of Medicine, Case Western Reserve University, Cleveland, OH 44106.
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D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000737:chemistry*; D000345:Affinity Labels; D000818:Animals; D001665:Binding Sites; D009130:Muscle, Smooth / Q000737:chemistry*; D009218:Myosins / Q000737:chemistry* / Q000378:metabolism; D009994:Osmolar Concentration; D010777:Photochemistry; D011189:Potassium Chloride; D011487:Protein Conformation; D014422:Turkeys; D062367:ortho-Aminobenzoates / Q000737:chemistry*
D000345:Affinity Labels; D062367:ortho-Aminobenzoates; D011189:Potassium Chloride; D000255:Adenosine Triphosphate; D009218:Myosins