Purification of an 80 kDa Ca²⁺-Dependent Actin-Modulating Protein, Which Severs Actin Filaments, from Bovine Adrenal Medulla

Abstract

A protein with a molecular weight of 80 kDa, which binds Ca²⁺-dependently to actin, was purified chromatographically from bovine adrenal medulla by using Sephacryl S-300, DEAE-Sepharose, actin-DNase I Sepharose, and Sephacryl S-200. This protein was retained on an actin-DNase I affinity column only in the presence of Ca²⁺, and could be eluted from this column by EGTA. The 80 kDa protein is a monomer and binds to G-actin in a Ca²⁺-dependent manner at an equimolar ratio. It caused fragmentation of actin filaments at more than 4×10^-7 M free Ca²⁺ concentration, as determined by low-shear viscometry and electron microscopy. Saturating amounts of tropomyosin showed a slight protective effect on the fragmentation of actin filaments by the 80 kDa protein. Considering the mode of action on actin filaments, the 80 kDa protein reported here seems to be a gelsolin-like protein. Gel electrophoresis of this protein revealed changes in mobility depending upon the concentration of Ca²⁺. This result also indicates that the 80 kDa protein itself is a Ca²⁺-binding protein.