Identification of Actin Surface Interacting with Myosin During the Actin-Myosin Sliding
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We constructed several mutant actin genes from the Dictyostelium actin 15 gene by the site-directed mutagenesis. Mutations were designed to change acidic residues in actin subdomain 1 to histidine residues. Amino acid replacements were: D1H (single replacement of Aspl to His), D4H, D1H/D4H (double replacements of Asp1 and Asp4 to histidine), D1H/E3H/D4H (triple replacements of Aspl, Glu3 and Asp4 to histidine), D24H/D25H, E99H/E100H, E360H/E361H, and D363H/E364H. Mutant genes were then expressed in Dictyostelium cells. In vitro motility assays were carried out for purified actins to see whether the mutations affect sliding motion of actin filaments driven by HMM. The assays showed that replacement of N-terminal acidic residues inhibited the sliding. Replacement of D24/D25 and E99/E100 also resulted in inhibition of the sliding motion. However, replacement of acidic residues at the C-terminal cluster E360/E361/D363/E363 did not resulted in loss of motility.
KeywordsATPase Activity Amino Acid Replacement Motility Assay Dictyostelium Cell Mutant Actin
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