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. 1989 Aug 1;109(2):585–591. doi: 10.1083/jcb.109.2.585

Purification and characterization of an Acanthamoeba nuclear actin- binding protein

PMCID: PMC2115709  PMID: 2760108

Abstract

Immunolocalization of monoclonal antibodies to Acanthamoeba myosin I showed a cross-reactive protein in nuclei (Hagen, S. J., D. P. Kiehart, D. A. Kaiser, and T. D. Pollard. 1986. J. Cell Biol. 103:2121-2128). This protein is antigenically related to myosin I in that nine monoclonal antibodies and three polyclonal antibodies are cross- reactive. However, studies with affinity-purified antibodies and two- dimensional peptide maps show that the protein is not a proteolytic product of myosin I. We have used cell fractionation and column chromatography to purify this protein. It is a dimer of 34-kD polypeptides with a Stokes' radius of 4 nm. A polyclonal antisera generated against the purified protein confirms the nuclear localization seen with the cross-reactive monoclonal antibodies. The 34- kD protein binds actin filaments in an ATP-insensitive manner with a Kd of approximately 0.25 microM without cross-linking, severing, or capping. No ATPase activity was detected in the presence or absence of actin. It also binds to DNA. These unique properties suggest we have discovered a new class of actin-binding protein. We have given this protein the name NAB for "nuclear actin-binding" protein.

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Selected References

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  1. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  2. Bremer J. W., Busch H., Yeoman L. C. Evidence for a species of nuclear actin distinct from cytoplasmic and muscles actins. Biochemistry. 1981 Mar 31;20(7):2013–2017. doi: 10.1021/bi00510a042. [DOI] [PubMed] [Google Scholar]
  3. Clark T. G., Merriam R. W. Diffusible and bound actin nuclei of Xenopus laevis oocytes. Cell. 1977 Dec;12(4):883–891. doi: 10.1016/0092-8674(77)90152-0. [DOI] [PubMed] [Google Scholar]
  4. Cooper J. A., Blum J. D., Pollard T. D. Acanthamoeba castellanii capping protein: properties, mechanism of action, immunologic cross-reactivity, and localization. J Cell Biol. 1984 Jul;99(1 Pt 1):217–225. doi: 10.1083/jcb.99.1.217. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cooper J. A., Blum J. D., Williams R. C., Jr, Pollard T. D. Purification and characterization of actophorin, a new 15,000-dalton actin-binding protein from Acanthamoeba castellanii. J Biol Chem. 1986 Jan 5;261(1):477–485. [PubMed] [Google Scholar]
  6. Elder J. H., Pickett R. A., 2nd, Hampton J., Lerner R. A. Radioiodination of proteins in single polyacrylamide gel slices. Tryptic peptide analysis of all the major members of complex multicomponent systems using microgram quantities of total protein. J Biol Chem. 1977 Sep 25;252(18):6510–6515. [PubMed] [Google Scholar]
  7. Fujiwara K., Pollard T. D. Fluorescent antibody localization of myosin in the cytoplasm, cleavage furrow, and mitotic spindle of human cells. J Cell Biol. 1976 Dec;71(3):848–875. doi: 10.1083/jcb.71.3.848. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Gadasi H., Korn E. D. Evidence for differential intracellular localization of the Acanthamoeba myosin isoenzymes. Nature. 1980 Jul 31;286(5772):452–456. doi: 10.1038/286452a0. [DOI] [PubMed] [Google Scholar]
  9. Hagen S. J., Kiehart D. P., Kaiser D. A., Pollard T. D. Characterization of monoclonal antibodies to Acanthamoeba myosin-I that cross-react with both myosin-II and low molecular mass nuclear proteins. J Cell Biol. 1986 Dec;103(6 Pt 1):2121–2128. doi: 10.1083/jcb.103.6.2121. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Isenberg G., Aebi U., Pollard T. D. An actin-binding protein from Acanthamoeba regulates actin filament polymerization and interactions. Nature. 1980 Dec 4;288(5790):455–459. doi: 10.1038/288455a0. [DOI] [PubMed] [Google Scholar]
  11. Jung G., Korn E. D., Hammer J. A., 3rd The heavy chain of Acanthamoeba myosin IB is a fusion of myosin-like and non-myosin-like sequences. Proc Natl Acad Sci U S A. 1987 Oct;84(19):6720–6724. doi: 10.1073/pnas.84.19.6720. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. 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]
  13. Korn E. D., Hammer J. A., 3rd Myosins of nonmuscle cells. Annu Rev Biophys Biophys Chem. 1988;17:23–45. doi: 10.1146/annurev.bb.17.060188.000323. [DOI] [PubMed] [Google Scholar]
  14. Kumar A., Raziuddin, Finlay T. H., Thomas J. O., Szer W. Isolation of a minor species of actin from the nuclei of Acanthamoeba castellanii. Biochemistry. 1984 Dec 18;23(26):6753–6757. doi: 10.1021/bi00321a072. [DOI] [PubMed] [Google Scholar]
  15. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  16. Lynch T. J., Albanesi J. P., Korn E. D., Robinson E. A., Bowers B., Fujisaki H. ATPase activities and actin-binding properties of subfragments of Acanthamoeba myosin IA. J Biol Chem. 1986 Dec 25;261(36):17156–17162. [PubMed] [Google Scholar]
  17. MacLean-Fletcher S., Pollard T. D. Identification of a factor in conventional muscle actin preparations which inhibits actin filament self-association. Biochem Biophys Res Commun. 1980 Sep 16;96(1):18–27. doi: 10.1016/0006-291x(80)91175-4. [DOI] [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. McKay R. D. Binding of a simian virus 40 T antigen-related protein to DNA. J Mol Biol. 1981 Jan 25;145(3):471–488. doi: 10.1016/0022-2836(81)90540-4. [DOI] [PubMed] [Google Scholar]
  20. Nishida E., Iida K., Yonezawa N., Koyasu S., Yahara I., Sakai H. Cofilin is a component of intranuclear and cytoplasmic actin rods induced in cultured cells. Proc Natl Acad Sci U S A. 1987 Aug;84(15):5262–5266. doi: 10.1073/pnas.84.15.5262. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Pollard T. D., Cooper J. A. Actin and actin-binding proteins. A critical evaluation of mechanisms and functions. Annu Rev Biochem. 1986;55:987–1035. doi: 10.1146/annurev.bi.55.070186.005011. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Pollard T. D. Purification of a high molecular weight actin filament gelation protein from Acanthamoeba that shares antigenic determinants with vertebrate spectrins. J Cell Biol. 1984 Dec;99(6):1970–1980. doi: 10.1083/jcb.99.6.1970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. 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]
  25. Rimm D. L., Sinard J. H., Pollard T. D. Location of the head-tail junction of myosin. J Cell Biol. 1989 May;108(5):1783–1789. doi: 10.1083/jcb.108.5.1783. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Stossel T. P., Chaponnier C., Ezzell R. M., Hartwig J. H., Janmey P. A., Kwiatkowski D. J., Lind S. E., Smith D. B., Southwick F. S., Yin H. L. Nonmuscle actin-binding proteins. Annu Rev Cell Biol. 1985;1:353–402. doi: 10.1146/annurev.cb.01.110185.002033. [DOI] [PubMed] [Google Scholar]
  27. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Warrick H. M., Spudich J. A. Myosin structure and function in cell motility. Annu Rev Cell Biol. 1987;3:379–421. doi: 10.1146/annurev.cb.03.110187.002115. [DOI] [PubMed] [Google Scholar]
  29. Wray W., Boulikas T., Wray V. P., Hancock R. Silver staining of proteins in polyacrylamide gels. Anal Biochem. 1981 Nov 15;118(1):197–203. doi: 10.1016/0003-2697(81)90179-2. [DOI] [PubMed] [Google Scholar]

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