Isolation and characterization of a Ca2+-activated actin-modulating protein from obliquely striated muscle

Summary An actin-modulating protein has been isolated from the obliquely striated body wall muscle of the earthwormLumbricus terrestris. The isolation procedure included extraction in the absence of Ca²⁺ with subsequent ammonium sulfate fractionation followed by ion exchange chromatography and gel filtration. The purified modulator preparation appears on SDS-PAGE as a doublet of bands with molecular weights of 43 and 45 kDa. Both separately isolated components exhibit the same actin-modulating properties and therefore probably represent two isoforms. Substoichiometric amounts of modulator sever actin filaments and effectively promote nucleation of actin polymerization, which results in the formation of short actin filaments. Both effets are completely dependent on the presence of Ca²⁺ and activation of the modulator occurs in a narrow range of free Ca²⁺ concentrations around 10^–6 mmol l^–1. The modulator increases the critical concentration for actin polymerization, indicating that it binds to the fast polymerizing end of the actin filaments. The modulator forms a stoichiometric complex with two actin molecules whereby its ability to sever actin filaments is lost.The properties of the earthworm actin modulator are discussed in comparison with similar actinassociated proteins.Abbreviations BSA bovine serum albumin - DTE dithioerythritol - EWAM earthworm actin modulator - IEF isoelectric focusing - PAGE polyacrylamide gel electrophoresis - PMSF phenylmethylsulfonyl fluoride - SDS sodium dodecyl sulfateDedicated to Professor Dr. K.-E. Wohlfarth-Bottermann, Bonn, on the occasion of his 65th birthday