Antibody Directed against the 142–148 Sequence of the Myosin Heavy Chain Interferes with Myosin-Actin Interaction

Mary Dan-Goor, Andras Muhlrad*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

12 Scopus citations

Abstract

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.

Original languageEnglish
Pages (from-to)400-405
Number of pages6
JournalBiochemistry
Volume30
Issue number2
DOIs
StatePublished - 1 May 1991

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