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. 1984 Sep 1;99(3):1024–1033. doi: 10.1083/jcb.99.3.1024

Inhibition of acanthamoeba actomyosin-II ATPase activity and mechanochemical function by specific monoclonal antibodies

PMCID: PMC2113385  PMID: 6206075

Abstract

Monoclonal and polyclonal antibodies that bind to myosin-II were tested for their ability to inhibit myosin ATPase activity, actomyosin ATPase activity, and contraction of cytoplasmic extracts. Numerous antibodies specifically inhibit the actin activated Mg++-ATPase activity of myosin- II in a dose-dependent fashion, but none blocked the ATPase activity of myosin alone. Control antibodies that do not bind to myosin-II and several specific antibodies that do bind have no effect on the actomyosin-II ATPase activity. In most cases, the saturation of a single antigenic site on the myosin-II heavy chain is sufficient for maximal inhibition of function. Numerous monoclonal antibodies also block the contraction of gelled extracts of Acanthamoeba cytoplasm. No polyclonal antibodies tested inhibited ATPase activity or gel contraction. As expected, most antibodies that block actin-activated ATPase activity also block gel contraction. Exceptions were three antibodies M2.2, -15, and -17, that appear to uncouple the ATPase activity from gel contraction: they block gel contraction without influencing ATPase activity. The mechanisms of inhibition of myosin function depends on the location of the antibody-binding sites. Those inhibitory antibodies that bind to the myosin-II heads presumably block actin binding or essential conformational changes in the myosin heads. A subset of the antibodies that bind to the proximal end of the myosin- II tail inhibit actomyosin-II ATPase activity and gel contraction. Although this part of the molecule is presumably some distance from the ATP and actin-binding sites, these antibody effects suggest that structural domains in this region are directly involved with or coupled to catalysis and energy transduction. A subset of the antibodies that bind to the tip of the myosin-II tail appear to inhibit ATPase activity and contraction through their inhibition of filament formation. They provide strong evidence for a substantial enhancement of the ATPase activity of myosin molecules in filamentous form and suggest that the myosin filaments may be required for cell motility.

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Selected References

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  1. Adelstein R. S., Eisenberg E. Regulation and kinetics of the actin-myosin-ATP interaction. Annu Rev Biochem. 1980;49:921–956. doi: 10.1146/annurev.bi.49.070180.004421. [DOI] [PubMed] [Google Scholar]
  2. Collins J. H., Korn E. D. Actin activation of Ca2+-sensitive Mg2+-ATPase activity of Acanthamoeba myosin II is enhanced by dephosphorylation of its heavy chains. J Biol Chem. 1980 Sep 10;255(17):8011–8014. [PubMed] [Google Scholar]
  3. Collins J. H., Korn E. D. Purification and characterization of actin-activatable, Ca2+-sensitive myosin II from Acanthamoeba. J Biol Chem. 1981 Mar 10;256(5):2586–2595. [PubMed] [Google Scholar]
  4. Craig R., Smith R., Kendrick-Jones J. Light-chain phosphorylation controls the conformation of vertebrate non-muscle and smooth muscle myosin molecules. 1983 Mar 31-Apr 6Nature. 302(5907):436–439. doi: 10.1038/302436a0. [DOI] [PubMed] [Google Scholar]
  5. Goody R. S., Holmes K. C. Cross-bridges and the mechanism of muscle contraction. Biochim Biophys Acta. 1983 Apr 15;726(1):13–39. doi: 10.1016/0304-4173(83)90009-5. [DOI] [PubMed] [Google Scholar]
  6. Hardwicke P. M., Wallimann T., Szent-Györgyi A. G. Light-chain movement and regulation in scallop myosin. Nature. 1983 Feb 10;301(5900):478–482. doi: 10.1038/301478a0. [DOI] [PubMed] [Google Scholar]
  7. Harrington W. F. On the origin of the contractile force in skeletal muscle. Proc Natl Acad Sci U S A. 1979 Oct;76(10):5066–5070. doi: 10.1073/pnas.76.10.5066. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Huxley H. E. Structural difference between resting and rigor muscle; evidence from intensity changes in the lowangle equatorial x-ray diagram. J Mol Biol. 1968 Nov 14;37(3):507–520. doi: 10.1016/0022-2836(68)90118-6. [DOI] [PubMed] [Google Scholar]
  9. Kiehart D. P., Kaiser D. A., Pollard T. D. Direct localization of monoclonal antibody-binding sites on Acanthamoeba myosin-II and inhibition of filament formation by antibodies that bind to specific sites on the myosin-II tail. J Cell Biol. 1984 Sep;99(3):1015–1023. doi: 10.1083/jcb.99.3.1015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Kiehart D. P., Kaiser D. A., Pollard T. D. Monoclonal antibodies demonstrate limited structural homology between myosin isozymes from Acanthamoeba. J Cell Biol. 1984 Sep;99(3):1002–1014. doi: 10.1083/jcb.99.3.1002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Kiehart D. P., Mabuchi I., Inoué S. Evidence that myosin does not contribute to force production in chromosome movement. J Cell Biol. 1982 Jul;94(1):165–178. doi: 10.1083/jcb.94.1.165. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Korn E. D. Biochemistry of actomyosin-dependent cell motility (a review). Proc Natl Acad Sci U S A. 1978 Feb;75(2):588–599. doi: 10.1073/pnas.75.2.588. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Kuznicki J., Albanesi J. P., Côté G. P., Korn E. D. Supramolecular regulation of the actin-activated ATPase activity of filaments of Acanthamoeba Myosin II. J Biol Chem. 1983 May 25;258(10):6011–6014. [PubMed] [Google Scholar]
  14. Lowey S., Slayter H. S., Weeds A. G., Baker H. Substructure of the myosin molecule. I. Subfragments of myosin by enzymic degradation. J Mol Biol. 1969 May 28;42(1):1–29. doi: 10.1016/0022-2836(69)90483-5. [DOI] [PubMed] [Google Scholar]
  15. Mabuchi I., Okuno M. The effect of myosin antibody on the division of starfish blastomeres. J Cell Biol. 1977 Jul;74(1):251–263. doi: 10.1083/jcb.74.1.251. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. MacLean-Fletcher S. D., Pollard T. D. Viscometric analysis of the gelation of Acanthamoeba extracts and purification of two gelation factors. J Cell Biol. 1980 May;85(2):414–428. doi: 10.1083/jcb.85.2.414. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Maruta H., Korn E. D. Direct photoaffinity labeling by nucleotides of the apparent catalytic site on the heavy chains of smooth muscle and Acanthamoeba myosins. J Biol Chem. 1981 Jan 10;256(1):499–502. [PubMed] [Google Scholar]
  18. Maruta H., Korn E. D. Purification from Acanthamoeba castellanii of proteins that induce gelation and syneresis of F-actin. J Biol Chem. 1977 Jan 10;252(1):399–402. [PubMed] [Google Scholar]
  19. McLachlan A. D., Karn J. Periodic charge distributions in the myosin rod amino acid sequence match cross-bridge spacings in muscle. Nature. 1982 Sep 16;299(5880):226–231. doi: 10.1038/299226a0. [DOI] [PubMed] [Google Scholar]
  20. McLachlan A. D., Karn J. Periodic features in the amino acid sequence of nematode myosin rod. J Mol Biol. 1983 Mar 15;164(4):605–626. doi: 10.1016/0022-2836(83)90053-0. [DOI] [PubMed] [Google Scholar]
  21. PORTER R. R. The hydrolysis of rabbit y-globulin and antibodies with crystalline papain. Biochem J. 1959 Sep;73:119–126. doi: 10.1042/bj0730119. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Pollard T. D. Electron microscopy of synthetic myosin filaments. Evidence for cross-bridge. Flexibility and copolymer formation. J Cell Biol. 1975 Oct;67(1):93–104. doi: 10.1083/jcb.67.1.93. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Pollard T. D., Korn E. D. Acanthamoeba myosin. I. Isolation from Acanthamoeba castellanii of an enzyme similar to muscle myosin. J Biol Chem. 1973 Jul 10;248(13):4682–4690. [PubMed] [Google Scholar]
  24. Pollard T. D. Myosin purification and characterization. Methods Cell Biol. 1982;24:333–371. doi: 10.1016/s0091-679x(08)60665-2. [DOI] [PubMed] [Google Scholar]
  25. Pollard T. D. Purification of a calcium-sensitive actin gelation protein from Acanthamoeba. J Biol Chem. 1981 Jul 25;256(14):7666–7670. [PubMed] [Google Scholar]
  26. Pollard T. D., Stafford W. F., Porter M. E. Characterization of a second myosin from Acanthamoeba castellanii. J Biol Chem. 1978 Jul 10;253(13):4798–4808. [PubMed] [Google Scholar]
  27. Pollard T. D. Structure and polymerization of Acanthamoeba myosin-II filaments. J Cell Biol. 1982 Dec;95(3):816–825. doi: 10.1083/jcb.95.3.816. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Pollard T. D. The role of actin in the temperature-dependent gelation and contraction of extracts of Acanthamoeba. J Cell Biol. 1976 Mar;68(3):579–601. doi: 10.1083/jcb.68.3.579. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Scholey J. M., Taylor K. A., Kendrick-Jones J. Regulation of non-muscle myosin assembly by calmodulin-dependent light chain kinase. Nature. 1980 Sep 18;287(5779):233–235. doi: 10.1038/287233a0. [DOI] [PubMed] [Google Scholar]
  30. Sheetz M. P., Spudich J. A. Movement of myosin-coated fluorescent beads on actin cables in vitro. Nature. 1983 May 5;303(5912):31–35. doi: 10.1038/303031a0. [DOI] [PubMed] [Google Scholar]
  31. Taylor D. L., Condeelis J. S. Cytoplasmic structure and contractility in amoeboid cells. Int Rev Cytol. 1979;56:57–144. doi: 10.1016/s0074-7696(08)61821-5. [DOI] [PubMed] [Google Scholar]
  32. Taylor E. W. Mechanism of actomyosin ATPase and the problem of muscle contraction. CRC Crit Rev Biochem. 1979;6(2):103–164. doi: 10.3109/10409237909102562. [DOI] [PubMed] [Google Scholar]
  33. Thomas D. D., Seidel J. C., Hyde J. S., Gergely J. Motion of subfragment-1 in myosin and its supramolecular complexes: saturation transfer electron paramagnetic resonance. Proc Natl Acad Sci U S A. 1975 May;72(5):1729–1733. doi: 10.1073/pnas.72.5.1729. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Trybus K. M., Huiatt T. W., Lowey S. A bent monomeric conformation of myosin from smooth muscle. Proc Natl Acad Sci U S A. 1982 Oct;79(20):6151–6155. doi: 10.1073/pnas.79.20.6151. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Tsong T. Y., Himmelfarb S., Harrington W. F. Stability and melting kinetics of structural domains in the myosin rod. J Mol Biol. 1983 Mar 5;164(3):431–450. doi: 10.1016/0022-2836(83)90060-8. [DOI] [PubMed] [Google Scholar]
  36. Vibert P., Craig R. Three-dimensional reconstruction of thin filaments decorated with a Ca2+-regulated myosin. J Mol Biol. 1982 May 15;157(2):299–319. doi: 10.1016/0022-2836(82)90236-4. [DOI] [PubMed] [Google Scholar]
  37. Yano M., Yamamoto Y., Shimizu H. An actomyosin motor. Nature. 1982 Oct 7;299(5883):557–559. doi: 10.1038/299557a0. [DOI] [PubMed] [Google Scholar]

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