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Published on: August 28, 2012
Purification and characterization of an Acanthamoeba nuclear actin-binding protein
1Department of Cell Biology and Anatomy, Johns Hopkins School of Medicine, Baltimore, Maryland 21205.
Insights
Researchers discovered a novel nuclear actin-binding protein (NAB) in Acanthamoeba. This protein, a dimer of 34-kD polypeptides, binds actin filaments and DNA, suggesting a new class of actin-binding proteins.
Area of Science:
- Cell Biology
- Biochemistry
- Molecular Biology
Background:
- Monoclonal antibodies to Acanthamoeba myosin I identified a cross-reactive nuclear protein.
- This protein is antigenically related to myosin I but not a proteolytic product.
Purpose of the Study:
- To purify and characterize the novel nuclear protein.
- To determine the protein's biochemical properties and potential functions.
Main Methods:
- Cell fractionation and column chromatography were used for protein purification.
- Characterization involved determining polypeptide size, Stokes' radius, and actin/DNA binding assays.
- Antisera generation confirmed nuclear localization.
Main Results:
- A dimer of 34-kD polypeptides was purified, with a Stokes' radius of 4 nm.
- The protein binds actin filaments ATP-insensitively (Kd ≈ 0.25 µM) without cross-linking, severing, or capping.
- No ATPase activity was detected, and the protein also binds DNA.
- Polyclonal antisera confirmed nuclear localization.
Conclusions:
- A new class of actin-binding protein, named nuclear actin-binding (NAB) protein, has been identified.
- NAB exhibits unique properties, including actin and DNA binding, suggesting novel cellular roles.
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.
