Cross-linking of the 25- and 20-kilodalton fragments of skeletal myosin subfragment 1 by a bifunctional ATP analogue.
The bifunctional photoreactive ATP analogue azidonitrobenzoyl-8-azido-ATP (ANB-8-N3-ATP) was synthesized. This ATP analogue carriers photoreactive azido groups at the eighth position of the adenine ring and at the 3' position of ribose. Photolysis of this analogue in the presence of skeletal muscle alpha-chymotryptic subfragment 1 (S-1) resulted in a new 120-kDa band, while photolysis in the presence of the tryptic S-1 produced a new 45-kDa band. The 45-kDa peptide was shown to be combined with the 25-kDa N-terminal and 20-kDa C-terminal fragments since it was labeled with a monoclonal antibody specific for the N-terminal 25-kDa segment of the S-1 heavy chain, and it was also found to retain the fluorescence of (iodoacetamido)fluorescein attached specifically to the SH-1 thiol of the C-terminal 20-kDa segment. These results indicate that the 25- and 20-kDa peptides are in close contact with the ATPase active site.
Maruta
S
S
Department of Physiology and Biophysics, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106.
Burke
M
M
Ikebe
M
M
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000494:pharmacology; D000818:Animals; D001386:Azides / Q000494:pharmacology*; D001665:Binding Sites; D003432:Cross-Linking Reagents / Q000494:pharmacology* / Q000528:radiation effects; D015394:Molecular Structure; D008970:Molecular Weight; D015879:Myosin Subfragments / Q000378:metabolism*; D010777:Photochemistry
D001386:Azides; D003432:Cross-Linking Reagents; D015879:Myosin Subfragments; C066960:5-azido-2-nitrobenzoyl-8-azido ATP; D000255:Adenosine Triphosphate