Brevin and vitamin D binding protein: comparison of the effects of two serum proteins on actin assembly and disassembly.

Actin depolymerizing activity in serum can be attributed to the two proteins brevin and vitamin D binding protein (DBP). To investigate their mechanisms of action, we used a number of techniques, including procedures involving the fluorescent pyrene-labeled actin probe, to compare the interaction of the two proteins with G- and F-actin in vitro. With a fluorescence enhancement assay, we determined that brevin forms a 1:2 complex and DBP forms a 1:1 complex with pyrene-G-actin. We also found that both proteins reduce the viscosity of F-actin measured with high-shear and low-shear viscometers, with brevin effective at much lower concentrations than DBP. In polymerization experiments, brevin inhibits filament elongation at substoichiometric levels by inhibiting monomer addition at the barbed end but can also accelerate polymerization by nucleating assembly of filaments which grow from the pointed end. DBP does not nucleate filament assembly and inhibits filament elongation at either end only at near-stoichiometric levels. Brevin, but not DBP, accelerates disassembly of filaments diluted into a depolymerizing medium. This is consistent with the capability of brevin to sever preformed filaments associated with erythrocyte membranes and to increase the number of filament ends as estimated by a cytochalasin binding assay. In steady-state experiments involving the use of pyrene-actin, brevin produces only a small increase in the apparent monomer concentration when the critical concentrations at the two ends of the filaments are the same (i.e., in 0.1 M KCl). However, when the critical concentration at the pointed end is higher than that at the barbed end (i.e., in 2 mM MgCl2), low molar ratios of brevin sharply increase the monomer concentration to the critical concentration of the pointed end. This allows substoichiometric amounts of brevin to completely depolymerize filaments when the total actin concentration is at or below that of the pointed end. In contrast to brevin, DBP increases the amount of nonfilamentous actin in a stoichiometric and dose-dependent manner regardless of the nature of the salt in the medium. We conclude from this study that brevin is similar in its mechanism of action to other proteins known to bind to the barbed end of filaments and that DBP is related in its action to proteins that complex monomers and prevent them from participating in the polymerization process.