Plasma actin depolymerizing factor has both calcium-dependent and calcium-independent effects on actin

Abstract

The effects of pig plasma actin depolymerizing factor (ADF) on both G-actin polymerization and F-actin fragmentation were examined using rabbit skeletal muscle actin labeled with N-(1-pyrenyl)iodoacetamide, a sensitive fluorescence probe for monomer to filament interconversion. Fluorescence data were compared with results obtained by viscometry and by difference absorption measurements at 232 nm. Plasma ADF nucleates actin filament assembly in a Ca2+-dependent manner; actin polymerization rates are enhanced at greater than 10-6 M Ca2+. The Ca concentration dependence of this effect, showing a shift in ADF nucleating capacity between 10-6 and 10-7 M Ca2+, is that expected for an intracellular regulatory effect, but in plasma, the protein would always be saturated with Ca2+. Although the rate of polymerization is markedly enhanced in the presence of Ca2+ ions, the extent of polymerization (as determined by the amplitude of the fluorescence change or the specific viscosity) is reduced in the presence of ADF and shows little or no Ca2+ dependence. The critical concentration of actin monomers is increased in the presence of ADF whether Ca is present or not. When ADF is added to F-actin, there is an immediate fall in fluorescence. This conversion of filaments to monomers by ADF (as defined by the fluorescence changes) is unaffected by Ca concentration. Electron micrographs of F-actin treated with ADF show that the filaments are indeed shortened at both high and low Ca concentrations. Taken together, these observations are interpreted in terms of a model in which ADF has both Ca2+-sensitive and Ca2+-insensitive binding sites for actin.