Antibody directed against the 142-148 sequence of the myosin heavy chain interferes with myosin-actin interaction

• Published in: Biochemistry (Easton) 1991-01, Vol.30 (2), p.400-405
• Main Authors: Dan-Goor, Mary, Muhlrad, Andras
• Format: Article
• Language: English
• Subjects: actin; Actins - metabolism; Actomyosin - immunology; Actomyosin - physiology; Amino Acid Sequence; Analytical, structural and metabolic biochemistry; Animals; arginine; Biological and medical sciences; Blotting, Western; Contractile proteins; Epitopes; Fundamental and applied biological sciences. Psychology; Holoproteins; In Vitro Techniques; Molecular Sequence Data; Myosins - immunology; Myosins - metabolism; Peptide Fragments - chemical synthesis; Peptide Fragments - immunology; Peptide Fragments - metabolism; Protein Binding; Proteins; Rabbits
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00216a014

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.