Immunochemical probing of the functional role of the 238-246 and 567-574 sequences of myosin heavy chain.
Polyclonal site-directed peptide antibodies were raised against the 567-574 and 238-246 sequences of the rabbit skeletal muscle myosin heavy chain. These sequences, which are located in the subfragment 1 (S1) segment of myosin, have been implicated by former studies in actin and nucleotide binding of the molecule and in the communication between the two binding sites. The antibodies obtained from rabbit sera were found to be conformation-sensitive since they specifically reacted with S1 in solid-phase binding assay but not in Western blot. The binding of both antibodies to S1 was strongly inhibited by actin. The antibody against the 567-574 sequence, Ab567-574, moderately decreased the binding of S1 to actin filaments in rigor but not in the weakly-attached state, while Ab238-246 did not influence the binding of S1 to actin under either conditions. Both antibodies inhibited the actin activation of the MgATPase of S1 but did not affect MgATPase without actin or the Ca- and K(EDTA)-activated ATPase activities of S1. The sliding velocity of actin filaments in the in vitro motility assays were also reduced in the presence of the antibodies. Ab567-574 had especially strong inhibitory effect on the movement of actin filaments. The results indicate that the binding of antibodies may induce conformational changes, which propagate in the S1 structure, perturb the coupling between the binding sites and impair the motor function of myosin.
Blotnick
E
E
Department of Oral Biology, Hebrew University-Hadassah School of Dental Medicine, Jerusalem, Israel.
Miller
C
C
Groschel-Stewart
U
U
Muhlrad
A
A
D000199:Actins / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D004797:Enzyme-Linked Immunosorbent Assay; D018482:Muscle, Skeletal / Q000378:metabolism*; D018995:Myosin Heavy Chains / Q000737:chemistry / Q000378:metabolism*; D010449:Peptide Mapping; D011817:Rabbits
D000199:Actins; D018995:Myosin Heavy Chains