Fig. 4. Identification of a twitchin D2 peptide binding region on actin. (A) SDS-PAGE patterns of the digests of chicken fast skeletal actin by trypsin. Numbers above the gel represent the digestion time (h). M, molecular markers. (B) Isolation of the peptide that reacts to unphosphorylated TWD2-S from the digests of actin by reverse-phase high performance liquid chromatography with a TSKgel ODS-80T column (4.6 mmx15 cm). Numbers in the graph represent the fraction numbers collected in this experiment. Each fraction was subjected to a solid-phase binding assay with TWD2-S. Only Fraction 16 (asterisk) reacted with TWD2-S. The inset shows the results of the colorimetric binding assay. Note the change of color only for fraction 16. (C) Second reverse-phase chromatography for fraction 16. Fraction 16 was separated into three peaks and only fraction 16-2 reacted to TWD2-S. The inset shows the results of the colorimetric binding assay; only fraction 16-2 was positive. (D) The binding region of actin with the D2 peptide. Asterisks represent two aspartic acid residues essential for myosin-driven movement of thick filaments on actin-containing thin filaments in the presence of ATP and Ca²⁺. The sequence of the isolated peptide that reacted to TWD2-S is shown in red. The peptide synthesized and used for competitive binding assay with TWD2-S in the present study is represented in green. (E) Competitive binding assay between chicken fast skeletal actin and its synthetic peptide AGFAGDDAP, measured by solid-phase binding assays (see Materials and methods). TWD2-S was displaced from actin by increasing concentrations of the synthetic peptide. (F) A structural representation of actin. The TWD2-S binding region is shown in yellow.