Actobindin, a 9-Kda protein from Acanthamoeba castellanii, binds to phenylenedimaleimide cross-linked """"""""upper"""""""" dimers with substantially higher affinity than to 2 actin monomers, consistent with the hypothesis that actobindin blocks nucleation of actin polymerization by binding to native dimers. Under polymerizing conditions, actobindin catalytically generates a greater than stoichiometric concentration of nucleation incompetent dimers from native monomers. Using the rate constant of formation of nucleation incompetent dimers and the rate of addition of these dimers to F-actin, the polymerization kinetics of actin polymerization were successfully modeled over a wide range of concentrations of actin and actobindin. The biologic function of actobindin could be to inhibit nucleation of random filament formation in regions of high concentrations of unpolymerized actin while facilitating elongation of specific filaments in the same regions. Immunolocalization studies are consistent with this hypothesis because actobindin appears to be present in very high concentration in newly forming pseudopods where just such properties would seem to be useful. Other immunolocalization studies have shown that Acanthamoeba profilin I is mostly in cortical regions of the cell, associated with G-actin, while profilin II is mostly associated with the plasma membrane. Thus, profilin I may function mainly as an actin monomer binding protein and profilin II as a regulator of metabolism of membrane-associated PIP2. In other experiments, muscle and Acanthamoeba F-actin were found to behave very differently when added to extracts of Acanthamoeba. Muscle F-actin depolymerized more rapidly than when similarly diluted in buffer whereas amoeba F-actin was much more stable in extracts than in buffer. Also, muscle actin was selectively removed from co-polymers of muscle and amoeba actin when the co-polymers contained less than 50% muscle actin. These observations may have significance for experiments in which labeled muscle actin is used as an indicator of the behavior of actin in a non-muscle cell.
