Abstract
Aspartic acid residues in the N-terminal negative charge cluster of Dictyostelium actin were replaced with histidine residues by site-directed mutagenesis of the actin gene. The mutant actins were expressed in Dictyostelium cells and were purified to homogeneity by HPLC. Functional properties of the mutant actins were compared with those of the wild-type actin. (i) In vitro assays of the sliding movement of actin filaments driven by myosin showed that the movement was slowed by the mutations. (ii) The mutations diminished the actin-activated ATPase activity of myosin in such a way that the maximum turnover rate at infinite actin concentration (Vmax) dropped sharply without an appreciable change in the apparent affinity of actin and myosin (Kapp). These results indicate that the N-terminal negative charge cluster of actin is essential for the ATP-dependent actin-myosin interaction.