THE SPECIFIC NH2-TERMINAL SEQUENCE AC-EEED OF ALPHA-SMOOTH MUSCLE ACTIN PLAYS A ROLE IN POLYMERIZATION IN-VITRO AND IN-VIVO

The blocking effect of the NH2-terminal decapeptide of alpha-smooth mu scle (SM) actin AcEEED-STALVC on the binding of the specific monoclona l antibody anti-alpha SM-1 (Skalli, O., P, Ropraz, A. Trzeviak, G, Ben zonana, D. Gillessen, and G. Gabbiani. 1986, J. Cell Biol. 103:2787-27 96) 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 sal t conditions; the antibody binding did not hinder the incorporation of the actin antibody complex into the filaments. This action was not ex erted on skeletal muscle actin. After microinjection of the alpha-SM a ctin NH2-terminal decapeptide or of the epitopic peptide into cultured aortic smooth muscle cells, double immunofluorescence for alpha-SM ac tin and total actin showed a selective disappearance of alpha-SM actin staining, detectable at similar to 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 poly merization during the dynamic filament turnover in stress fibers. What ever the mechanism, this is the first evidence that the NH2 terminus o f an actin isoform plays a role in the regulation of polymerization in vitro and in vivo.