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The specific NH2-terminal sequence Ac-EEED of alpha-smooth muscle actin plays a role in polymerization in vitro and in vivo

Goethals, M.
Janmey, P. A.
Gabbiani, F.
Vandekerckhove, J.
Published in Journal of Cell Biology. 1995, vol. 130, no. 4, p. 887-895
Abstract The blocking effect of the NH2-terminal decapeptide of alpha-smooth muscle (SM) actin AcEEED-STALVC on the binding of the specific monoclonal antibody anti-alpha SM-1 (Skalli, O., P. Ropraz, A. Trzeviak, G. Benzonana, D. Gillessen, and G. Gabbiani. 1986. J. Cell Biol. 103:2787-2796) was compared with that of synthetic peptides modified by changing the acetyl group or by substituting an amino acid in positions 1 to 5. Using immunofluorescence and immunoblotting techniques, anti-alpha SM-1 binding was abolished by the native peptide and by peptides with a substitution in position 5, indicating that AcEEED is the epitope for anti-alpha SM-1. Incubation of anti-alpha SM-1 (or of its Fab fragment) with arterial SM actin increased polymerization in physiological salt conditions; the antibody binding did not hinder the incorporation of the actin antibody complex into the filaments. This action was not exerted on skeletal muscle actin. After microinjection of the alpha-SM actin NH2-terminal decapeptide or of the epitopic peptide into cultured aortic smooth muscle cells, double immunofluorescence for alpha-SM actin and total actin showed a selective disappearance of alpha-SM actin staining, detectable at approximately 30 min. When a control peptide (e.g. alpha-skeletal [SK] actin NH2-terminal peptide) was microinjected, this was not seen. This effect is compatible with the possibility that the epitopic peptide traps a protein involved in alpha-SM actin polymerization during the dynamic filament turnover in stress fibers. Whatever the mechanism, this is the first evidence that the NH2 terminus of an actin isoform plays a role in the regulation of polymerization in vitro and in vivo.
Keywords Actins/immunology/ metabolism/ultrastructureAmino Acid SequenceAnimalsAntibody SpecificityAorta/cytologyBinding, CompetitiveCentrifugationEpitopesImmunoblottingMicroinjectionsMicroscopy, ElectronMicroscopy, FluorescenceMolecular Sequence DataMuscle, Smooth, Vascular/ metabolismOligopeptides/immunology/ metabolismPeptide Fragments/immunologyPolymers/metabolismProtein BindingRatsRats, Wistar
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Other version: http://jcb.rupress.org/content/130/4/887.full.pdf
PMID: 7543902
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