Purification and characterization of an Acanthamoeba nuclear actin- binding protein

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.

[1]  T. Pollard,et al.  Location of the head-tail junction of myosin , 1989, The Journal of cell biology.

[2]  J. Hammer,et al.  Myosins of nonmuscle cells. , 1988, Annual review of biophysics and biophysical chemistry.

[3]  J. Hammer,et al.  The heavy chain of Acanthamoeba myosin IB is a fusion of myosin-like and non-myosin-like sequences. , 1987, Proceedings of the National Academy of Sciences of the United States of America.

[4]  S. Koyasu,et al.  Cofilin is a component of intranuclear and cytoplasmic actin rods induced in cultured cells. , 1987, Proceedings of the National Academy of Sciences of the United States of America.

[5]  J. Spudich,et al.  Myosin structure and function in cell motility. , 1987, Annual review of cell biology.

[6]  J. Albanesi,et al.  ATPase activities and actin-binding properties of subfragments of Acanthamoeba myosin IA. , 1986, The Journal of biological chemistry.

[7]  T. Pollard,et al.  Characterization of monoclonal antibodies to Acanthamoeba myosin-I that cross-react with both myosin-II and low molecular mass nuclear proteins , 1986, The Journal of cell biology.

[8]  T. Pollard,et al.  Purification and characterization of actophorin, a new 15,000-dalton actin-binding protein from Acanthamoeba castellanii. , 1986, The Journal of biological chemistry.

[9]  T D Pollard,et al.  Actin and actin-binding proteins. A critical evaluation of mechanisms and functions. , 1986, Annual review of biochemistry.

[10]  D. B. Smith,et al.  Nonmuscle actin-binding proteins. , 1985, Annual review of cell biology.

[11]  A. Kumar,et al.  Isolation of a minor species of actin from the nuclei of Acanthamoeba castellanii. , 1984, Biochemistry.

[12]  T. Pollard.  Purification of a high molecular weight actin filament gelation protein from Acanthamoeba that shares antigenic determinants with vertebrate spectrins , 1984, The Journal of cell biology.

[13]  T. Pollard,et al.  Monoclonal antibodies demonstrate limited structural homology between myosin isozymes from Acanthamoeba , 1984, The Journal of cell biology.

[14]  T. Pollard,et al.  Acanthamoeba castellanii capping protein: properties, mechanism of action, immunologic cross-reactivity, and localization , 1984, The Journal of cell biology.

[15]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[16]  J. Olmsted,et al.  Affinity purification of antibodies from diazotized paper blots of heterogeneous protein samples. , 1981, The Journal of biological chemistry.

[17]  J. Bremer,et al.  Evidence for a species of nuclear actin distinct from cytoplasmic and muscles actins. , 1981, Biochemistry.

[18]  R. McKay.  Binding of a simian virus 40 T antigen-related protein to DNA. , 1981, Journal of molecular biology.

[19]  T. Pollard,et al.  Identification of a factor in conventional muscle actin preparations which inhibits actin filament self-association. , 1980, Biochemical and biophysical research communications.

[20]  E. Korn,et al.  Evidence for differential intracellular localization of the Acanthamoeba myosin isoenzymes , 1980, Nature.

[21]  H. Towbin,et al.  Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. , 1979, Proceedings of the National Academy of Sciences of the United States of America.

[22]  T. Pollard,et al.  Characterization of a second myosin from Acanthamoeba castellanii. , 1978, The Journal of biological chemistry.

[23]  R. Merriam,et al.  Diffusible and bound actin in nuclei of xenopus laevis oocytes , 1977, Cell.

[24]  R. Lerner,et al.  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. , 1977, The Journal of biological chemistry.

[25]  E. Korn,et al.  Purification from Acanthamoeba castellanii of proteins that induce gelation and syneresis of F-actin. , 1977, The Journal of biological chemistry.

[26]  T. Pollard,et al.  Fluorescent antibody localization of myosin in the cytoplasm, cleavage furrow, and mitotic spindle of human cells , 1976, The Journal of cell biology.

[27]  M. M. Bradford.  A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. , 1976, Analytical biochemistry.

[28]  T. Pollard,et al.  Acanthamoeba myosin. I. Isolation from Acanthamoeba castellanii of an enzyme similar to muscle myosin. , 1973, The Journal of biological chemistry.

[29]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.