Actin filament structure probed with monoclonal antibodies.
The interaction of two monoclonal antibodies (mAbs) with actin has been characterized to map the epitopes defined by these mAbs and to determine the accessibility of these sites in the actin filament (F-actin). Both mAbs react specifically with actin in radioimmunoassays and Western blot assays, and by immunoprecipitation. The location of the epitopes within the primary structure of actin has been determined using limited proteolysis of actin and Western blots, or using immunoprecipitation of truncated actin fragments synthesized in a cell free translation assay. Both mAbs bind to the C-terminal fragment of actin (residues 68-375) produced by chymotrypsin cleavage. One epitope is further localized to a 9.9 kD peptide corresponding to residues 5-93. Therefore, the epitope defined by this mAb (2G11.4) lies between residues Lys68 and Glu93 of actin. The location of the other epitope was determined by immunoprecipitation of actin fragments synthesized in vitro. Removal of residues 356-365 from the C-terminus of actin completely abolished the binding of mAb 4E3.adl. Therefore, this mAb defines an epitope that involves residues between Trp356 and Ala365. The accessibility of these epitopes in native F-actin was determined with solution binding assays and characterized by immunoelectron microscopy. Monoclonal antibody 4E3.adl binds strongly to filaments, resulting in bundling or decoration of F-actin depending on the valency of the mAb, and indicating that the epitope is readily accessible in F-actin. In contrast, mAb 2G11.4 disrupts F-actin structure, resulting in the formation of an amorphous immunoprecipitate. These results place constraints on models of actin filament structure.
Moncman
C L
CL
Department of Pathology, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854.
Peng
I
I
Winkelmann
D A
DA
D000199:Actins / Q000737:chemistry / Q000276:immunology / Q000648:ultrastructure*; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D002999:Clone Cells; D003429:Cross Reactions; D000939:Epitopes / Q000032:analysis; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000032:analysis; D017386:Sequence Homology, Amino Acid
D000199:Actins; D000911:Antibodies, Monoclonal; D000939:Epitopes; D010446:Peptide Fragments