No 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.

Pig plasma gelsolin forms a ternary complex with monomeric actin in 0.1 mM CaC1, and a binary complex in EGTA (< 0.01 pM calcium), as shown by gel filtration and fluorescence changes when actin which had been treated with N-ethylmaleimide and 7-chloro-4-nitrobenzeno-2-oxa-1,3-diazole (NBD-actin) or with N-(1-pyreny1)iodoacetamide (PI-actin) binds to gelsolin. The fluorescence enhancement per actin molecule bound is similar in the binary and ternary complexes, but the affinity of gelsolin for labelled actin is very much greater in the presence of calcium. Furthermore, the formation of ternary complex exhibits strong positive cooperativity.Actin is probably a universal component of eukaryotic cells and participates in a variety of motile processes including cytoplasmic streaming, phagocytosis, morphological changes and cytokinesis. The dynamic nature of actin in non-muscle cells contrasts with its hhgly organised structure in striated muscle. Changes in the distribution of actin in cells and in its supramolecular organisation are effected by factors that promote the rapid assembly and dissociation of F-actin and its higher-order aggregates, such as isotropic gels and filamentous bundles. The intracellular processes by which F-actin is assembled from the profilactin pool and monomeric actin regenerated from filaments are not known, but 'actin capping' proteins are presumed to be involved. Capping proteins can be divided into two general classes: (a) those that bind to one or other end of a filament but do not sever filaments and (b) those that both sever filaments and cap their ends (for reviews see [5 -71). With few exceptions, the proteins so far characterised bind to the 'barbed' end of the actin filament (i.e. the preferred end for filament elongation; reviewed in [S]).Gelsolin was first isolated by Yin and Stossel from rabbit macrophages, in which it causes a calcium-sensitive dispersal of actin-containing gels [9]. It was established that gelsolin interacts with both monomeric and polymeric actin only in the presence of micromolar concentrations of calcium ions We previously reported that pig plasma gelsolin, in contrast to the macrophage protein, severs actin filaments both in the presence and absence of calcium ions [23]. It also nucleates actin polymerisation and its efficacy in this is greatly enhanced by micromolar concentrations of calcium ions. These observations suggest that gelsolin binds to both monomeric and polymeric forms of actin. The interaction with G-actin has been investigated here using gel filtration and fluorescence methods. Results show that pig plasma gelsolin forms a binary complex with monomeric actin in the absence of calcium and a ternary complex containing two actin molecules when calcium is present at micromolar concentrations. The affinity of gelsolin for actin is much greater in the presence than in the absence of calcium ions. Preliminary reports of some of these results [7, 241 showed apparent internal contradictions in determinations of the stoichiometry of actin-gelsolin i...

Plasma gelsolin caps actin filaments at their 'barbed' ends and severs them along their length. Capping has been demonstrated both by direct visualization using gold-labeled gelsolin and by inhibition of actin polymerization onto the barbed ends of fragments of the acrosomal process of Limulus sperm. Severing activity is demonstrated by the fact that actin filaments nucleated off acrosomal fragments are shortened or removed within a few seconds by added plasma gelsolin without any obvious disruption of the actin bundles in the acrosomal processes themselves.