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 have been examined by using rabbit skeletal muscle actin labeled with N-(1-pyrenyl)iodoacetamide, a sensitive fluorescent probe for monomer to filament interconversion. Fluorescence data have been 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 calcium 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 calcium 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 calcium 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 calcium concentration. Electron micrographs of F-actin treated with ADF show that the filaments are indeed shortened at both high and low calcium 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.

Human plasma actin‐depolymerizing factor

“…The regulation of ADF action by Ca2+ may be more complex in a tissue section than in vitro owing to as yet unknown factors which are not present in the purified system. This Ca2+ dependence of ADF action has been reported previously [lo, 15,161, though Harris and Weeds [12] have shown that certain activities of ADF are Ca'+-insensitive. These discrepancies may be in part explained by the use of different techniques to measure the same phenomena.…”

“…Moreover, we cannot exclude the possibility that plasma contains two pools of ADF, one Ca2+-sensitive, the other Ca2+-insensitive. Each could be extracted in different amounts by the different purifications procedures which have been used [12] (and this paper). Markey et al [32] have, in fact, purified on a DNase-actin column a 90-kDa protein from platelets, having actin-destabilizing activities some of which are Ca'+-sensitive and some of which are not.…”

Role of group-specific component (vitamin D binding protein) in clearance of actin from the circulation in the rabbit.

“…Plasma gelsolin displays both calcium-dependent and calcium-independent effects, and interacts with actin in a polymorphous fashion (49), with each molecule possessing two distinct actin-binding sites (48,(50)(51)(52)(53). At low plasma gelsolin:actin molar ratios, the actions of nucleating actin filament assembly, blocking the fast growing end of the filaments, and severing actin filaments, are all strongly affected by the presence of calcium (46,50,(52)(53)(54).…”

A Ca2+‐sensitive actin regulatory protein from smooth muscle

“…Over the past 5 years, Ca'+-dependent severing proteins have been identified in a number of non-muscle cells and tissues, including macrophage [2], platelets [3], blood plasma [4], microvillus [5], thyroid [6], Physarum [7,8], and Dictyostefium [9]; however, Ca2+-dependent severing and capping proteins have not been isolated from skeletal muscle.…”