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.
Rimm
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Department of Cell Biology and Anatomy, Johns Hopkins School of Medicine, Baltimore, Maryland 21205.
Pollard
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D000048:Acanthamoeba / Q000032:analysis*; D000199:Actins / Q000378:metabolism; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D002458:Cell Fractionation; D003429:Cross Reactions; D004247:DNA / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D008970:Molecular Weight; D009218:Myosins / Q000276:immunology; D009687:Nuclear Proteins / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D010449:Peptide Mapping; D011485:Protein Binding
D000199:Actins; D000911:Antibodies, Monoclonal; D009687:Nuclear Proteins; D004247:DNA; D009218:Myosins