Isolation and characterization of the N-terminal 23-kilodalton fragment of myosin subfragment 1.
The 23-kDa N-terminal tryptic fragment was isolated from the heavy chain of rabbit skeletal myosin subfragment 1 (S-1). The heavy-chain fragments were dissociated by guanidine hydrochloride following limited trypsinolysis, and the 23-kDa fragment was isolated by gel filtration and ion-exchange chromatography. Finally, the fragment was renatured by removing the denaturants. The CD spectrum of the renatured fragment shows the presence of ordered structure. The tryptophan fluorescence emission spectrum of the fragment is considerably shifted to the red upon adding guanidine hydrochloride which indicates that the tryptophans are located in relatively hydrophobic environments. The two 23-kDa tryptophans, unlike the rest of the S-1 tryptophans, are fully accessible to acrylamide as indicated by fluorescence quenching. The isolated 23-kDa fragment cosediments with F-actin in the ultracentrifuge and significantly increases the light scattering of actin in solution which indicates actin binding. The binding is rather tight (Kd = 0.1 microM) and ionic strength dependent (decreasing with increasing ionic strength). ATP, pyrophosphate, and ADP dissociate the 23-kDa-actin complex with decreasing effectiveness. The isolated 23-kDa fragment does not have ATPase activity; however, it inhibits the actin-activated ATPase activity of S-1 by competing presumably with S-1 for binding sites on actin. F-Actin binds to the 23-kDa fragment immobilized on the nitrocellulose membrane. The fragment was further cleaved, and one of the resulting peptides, containing the 130-204 stretch of residues, was found to bind actin on the nitrocellulose membrane, indicating that this region of the 23-kDa fragment participates in forming an actin binding site.
Muhlrad
A
A
Cardiovascular Research Institute, University of California, San Francisco 94143.
D000199:Actins / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000302:isolation & purification* / Q000378:metabolism; D000818:Animals; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D015151:Immunoblotting; D007700:Kinetics; D008970:Molecular Weight; D009132:Muscles / Q000378:metabolism; D015879:Myosin Subfragments; D009218:Myosins / Q000302:isolation & purification* / Q000378:metabolism; D010446:Peptide Fragments / Q000302:isolation & purification* / Q000378:metabolism; D011487:Protein Conformation; D011817:Rabbits; D014357:Trypsin
D000199:Actins; D015879:Myosin Subfragments; D010446:Peptide Fragments; D014357:Trypsin; D000251:Adenosine Triphosphatases; D009218:Myosins