Actin Filament Elongation Research Articles

Characterization of brevin, a serum protein that shortens actin filaments.

We have purified a protein from rabbit serum with a molecular weight of 90,000 that inhibits the polymerization of actin measured viscometrically and that we have named "brevin" (from the Latin breviare, to shorten). From the extent of purification, we estimate that this inhibitor constitutes 0.3% of the total protein in plasma and serum. Brevin is also present in sera from humans and rats. Almost all of the activity in blood is extracellular; only 1% is present in platelets or other cellular elements. Several lines of evidence indicate that brevin is the same protein as the factor described by Fagraeus and Norberg [Fagraeus, A. & Norberg, R. (1978) Curr. Top. Microbiol. Immunol. 82, 1-13] as an actin-depolymerizing factor (ADF). If ADF and brevin are identical, then "ADF" is an inappropriate name because we find that the protein shortens actin filaments without depolymerizing them. Thus, brevin causes little change in the critical concentration of monomeric actin, even though the inhibitor binds to monomeric actin complexed to DNase I-agarose. Binding of brevin to filaments was demonstrated by sedimenting the inhibitor with F-actin. From the amounts of actin and brevin sedimented, and from the lengths of filaments measured by electron microscopy, we calculated that the stoichiometry of binding is one brevin molecule per filament over a wide range of inhibitor concentrations. This stoichiometry suggests that brevin inhibits polymerization by binding at the end of elongating actin filaments, a mechanism similar to that proposed for several intracellular actin-binding proteins and for the cytochalasins. Its abundance suggests that brevin plays an important physiological role in serum, but one not directly concerned with intracellular motility. Therefore its relationship to cytoplasmic actin-binding proteins remains to be determined.

Association of actin with chromaffin granule membranes and the effect of cytochalasin B on the polarity of actin filament elongation

Membranes of chromaffin granules isolated from bovine adrenal medulla are shown to bind dihydrocytochalasin B with high affinity. These membranes also bound [ 3H]actin in a time- and Mg 2+-dependent manner and electron microscopy showed the presence of membrane-attached actin filaments following addition of exogenous actin. Binding of [ 3H]actin was partially inhibited by cytochalasin B. Electron microscopic analysis of heavy meromyosin-decorated, membrane-attached filaments showed terminally (end-on) attached filaments with both possible polarities (i.e., filaments with arrowheads pointing both towards and away from the membranes). Treatment of samples with cytochalasin B preferentially inhibited growth of filaments with their ‘barbed’ ends pointing away from membranes. These results are discussed with respect to the role of actin in secretory granule function and the mechanism of cytochalasin action.

Cytochalasins block actin filament elongation by binding to high affinity sites associated with F-actin.

We have found that addition of a small amount of filamentous muscle actin (F-actin) to a solution of globular actin (G-actin) in a low ionic strength medium resulted in rapid polymerization of the G-actin. This reaction was inhibited by substoichiometric levels of cytochalasins (relative potency: cytochalasin D greater than cytochalasin E approximately equal to cytochalasin B greater than dihydrocytochalasin B). Binding experiments show that F-actin, but not G-actin, contains high affinity binding sites for [3H]cytochalasin B; the number of sites detected was on the order of about one per actin filament (one site/500 actin monomers). This number remained unchanged when the actin (prepared by polymerization-depolymerization cycles) was further purified by ion exchange and gel filtration chromatography. Competitive displacement experiments showed that the relative affinity of several cytochalasins for these sites corresponds to their relative effectiveness in inhibiting actin polymerization induced by F-actin. These results suggest that actin filaments can accelerate the rate of polymerization of G-actin in low ionic strength medium by providing sites onto which actin monomers can be added, and that cytochalasins inhibit actin filament elongation by binding to high affinity sites located at the polymerization end of the filaments.