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Identification of Actin Surface Interacting with Myosin During the Actin-Myosin Sliding

  • Kazuo Sutoh
Chapter
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 332)

Abstract

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.

Keywords

ATPase Activity Amino Acid Replacement Motility Assay Dictyostelium Cell Mutant Actin 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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References

  1. 1.
    Kabsch, W., Mannhertz, H.G., Suck, D., Pai, E.F. & Holmes, K.C. Nature 347, 37–44 (1990).PubMedCrossRefGoogle Scholar
  2. 2.
    Sutoh, K. Biochemistry 21, 3654–3661 (1982).PubMedCrossRefGoogle Scholar
  3. 3.
    Sutoh, K. Biochemistry 22, 1579–1585 (1983).PubMedCrossRefGoogle Scholar
  4. 4.
    Sutoh, K., Ando, M., Sutoh, K. & Toyoshima, Y. Proc. Natl. Acad. Sci. USA 88, 7711–7714 (1991).PubMedCrossRefGoogle Scholar
  5. 5.
    Knecht, D., Cohen, S.M., Loomis, W.F. & Loodish, H.F. Mol. Cell Biol. 6, 3973–3983 (1986).PubMedGoogle Scholar
  6. 6.
    Kron, SJ. & Spudich, J.A. Proc. Natl Acad. Sci. USA 83, 6272–6276 (1986).PubMedCrossRefGoogle Scholar
  7. 7.
    Harada, Y., Noguchi, A., Kishino, A. & Yanagida, T. Nature 326, 805–808 (1987).PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 1993

Authors and Affiliations

  • Kazuo Sutoh
    • 1
  1. 1.Department of Pure and Applied SciencesCollege of Arts and Sciences University of TokyoKomabaJapan

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