Antibody directed against the 142-148 sequence of the myosin heavy chain interferes with myosin-actin interaction.
It has been reported recently that the isolated and renatured 23-kDa N-terminal fragment of rabbit skeletal muscle myosin binds tightly to F-actin in an ATP-dependent manner [Muhlrad, A. (1989) Biochemistry 28, 4002-4010]. The binding to actin is of electrostatic nature and may involve a positively charged cluster of residues on the 23-kDa fragment stretching from Arg-143 to Arg-147. An octapeptide containing this positive cluster was synthesized and coupled to BSA through a cysteine residue added to the N-terminus of the peptide. Polyclonal antibody was raised against the BSA-coupled peptide in rabbits which recognized the N-terminal 23-kDa fragment of rabbit skeletal myosin subfragment 1, and a peptide comprised of residues 122-204 of the 23K fragment in Western blots. The purified antibody [IgG and F(ab)] inhibited the actin-activated ATPase activity of S1 without affecting its Mg2(+)- and K+(EDTA)-modulated ATPase activity. Both IgG and F(ab) decreased the binding of S1 to F-actin in a sedimentation assay, and actin inhibited the binding of both IgG and F(ab) to S1 in a competitive binding assay. The cysteine thiol of the synthetic octapeptide was labeled by the fluorescent thiol reagent monobromobimane, and the labeled peptide was found to bind to actin in a sedimentation assay. The results support the possibility that the positively charged Arg-143 to Arg-147 stretch of residues on the 23-kDa fragment participates in actin binding of myosin and may represent an essential constituent of the actin-S1 interface.
Dan-Goor
M
M
Department of Oral Biology, Hebrew University-Hadassah School of Dental Medicine, Jerusalem, Israel.
Muhlrad
A
A
D000199:Actins / Q000378:metabolism; D000205:Actomyosin / Q000276:immunology / Q000502:physiology*; D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D000939:Epitopes; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D009218:Myosins / Q000276:immunology* / Q000378:metabolism; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000276:immunology / Q000378:metabolism; D011485:Protein Binding; D011817:Rabbits
D000199:Actins; D000939:Epitopes; D010446:Peptide Fragments; D000205:Actomyosin; D009218:Myosins