AN ACTIN-MODULATING PROTEIN FROM PHYSARUM-POLYCEPHALUM .1. ISOLATION AND PURIFICATION

  • 1 January 1981
    • journal article
    • research article
    • Vol. 23  (2) , 225-233
Abstract
High-speed centrifugation supernatants from slime mould plasmodia show considerable activities to inhibit the polymerization of actin as revealed by viscosity measurements. By following increasing inhibitory activities an actin modulating protein (AM-protein) was isolated and purified which affects the polymer state of actin. AM-protein has a peptide chain weight of 42,000 and is indistinguishable from actin by SDS(sodium dodecyl sulfate)-electrophoresis but can be distinguished by isoelectric focusing. Peptide maps from partial proteolytic digests of AM-protein and Physarum actin reveal no similarities excluding that AM-protein is a denatured or modified form of actin. The protein is isolated from crude extracts as a heterodimer with actin to which it strongly binds. This heterodimer affects the polymerization of large amounts of actin by inducing oligomeric or lowpolymer actin complexes and inhibiting the formation of long actin filaments. The AM-protein/actin heterodimer has only a slight effect on F-actin. It partially depolymerizes F-actin within several hours. By ion exchange chromatography in 8 M urea the AM-protein is separated from the actin. The purified AM-protein monomer is renatured and inhibits the polymerization of actin like the heterodimer but additionally, depolymerizes actin filaments very rapidly and effectively by breaking them into oligomer or low-polymer complexes. The addition of less than 1% AM-protein causes a decrease of the specific viscosity of an F-actin solution by 50%. The degree of polymerization inhibition and depolyerization of actin is strictly dependent on the amount of AM-protein added; a catalytic type of reaction between both proteins can be excluded.