Evidence that a 27-residue sequence is the actin-binding site of ABP-120.
Proteolysis experiments of ABP-120 from Dictyostelium discoideum have previously demonstrated that removal of residues 89-115 from a tryptic peptide which retains actin binding activity, abolishes actin binding (Bresnick, A. R., Warren, V., and Condeelis, J. (1990) J. Biol. Chem. 265, 9236-9240). Antibodies made against a synthetic peptide of this 27-amino acid sequence (27-mer) specifically immunoprecipitate native ABP-120 from Dictyostelium high speed supernatants, demonstrating that the 27-mer sequence is on the surface of the molecule as expected for an active site. ABP-120 is inhibited in its binding to F-actin by Fab' fragments of the anti-27-mer IgG. Half-maximal inhibition occurs at an approximate molar ratio of 7 Fab' fragments/ABP-120 monomer. Viscoelastic measurements indicate that ABP-120 forms fewer cross-links with F-actin in the presence of the 27-mer synthetic peptide than in its absence. In F-actin cosedimentation assays, the binding of ABP-120 to actin is inhibited by the 27-mer synthetic peptide. Furthermore, the 27-mer synthetic peptide cosediments with F-actin, whereas a control hydrophobic peptide and a synthetic peptide of residues 69-88 of ABP-120 do not cosediment with F-actin. These observations suggest a direct involvement of the 27-mer sequence in the actin binding activity of ABP-120.
Bresnick
A R
AR
Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, New York 10461.
Janmey
P A
PA
Condeelis
J
J
D000199:Actins / Q000378:metabolism*; D000595:Amino Acid Sequence; D000906:Antibodies / Q000302:isolation & purification; D001665:Binding Sites; D001666:Binding Sites, Antibody; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism*; D002846:Chromatography, Affinity; D004023:Dictyostelium / Q000378:metabolism*; D004548:Elasticity; D007140:Immunoglobulin Fab Fragments; D007700:Kinetics; D008840:Microfilament Proteins / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D014783:Viscosity
D000199:Actins; D000906:Antibodies; D002352:Carrier Proteins; D007140:Immunoglobulin Fab Fragments; D008840:Microfilament Proteins; C060680:abpC protein, Dictyostelium