Domain mapping of chicken gizzard caldesmon.
Limited proteolysis, affinity chromatography, and immunoblotting have been used to define the domains of chicken gizzard caldesmon, caldesmon120, that interact with calmodulin, F-actin, and a monoclonal antibody prepared using human platelet caldesmon. Treatment of caldesmon120 with chymotrypsin produces groups of fragments near 100, 80, 60, 38, and 20 kDa. Further digestion produces peptides between 40 and 50 kDa. The 100- and 80-kDa peptides cross-react with the monoclonal antibody; the smaller polypeptides do not. The kinetics of cleavage and the antibody studies indicate that the 38- and 80-kDa fragments are the two major pieces of the 120-kDa protein. The 38-kDa fragment, purified by high performance liquid chromatography, and several of its subfragments at 21 and 25 kDa sediment with F-actin, bind to calmodulin-Sepharose in the presence of Ca2+, and are displaced from F-actin by Ca2+-calmodulin. The 80-kDa fragments did not interact with F-actin or calmodulin. We have tentatively placed the 38-kDa fragment at the C-terminal using polyclonal antibodies selected against a beta-galactosidase-caldesmon120 fusion protein produced by a lambda gt11 lysogen. The 38-, 25-, and 21-kDa fragments cross-react with these antibodies; the 80- and 60-kDa fragments do not. Caldesmon77 from human platelets also cross-reacts with these selected antibodies. The results suggest that interacting calmodulin and F-actin binding sites are localized on a 38-kDa C-terminal fragment of caldesmon. The smallest subfragment of this peptide that binds to both F-actin and calmodulin-Sepharose is about 21 kDa. The monoclonal antibody epitope is tentatively localized near the N-terminal of caldesmon77 and must be within 50 kDa of the N-terminal on caldesmon120.
D000818:Animals; D000911:Antibodies, Monoclonal; D001665:Binding Sites; D002148:Calmodulin-Binding Proteins / Q000032:analysis*; D002645:Chickens; D002846:Chromatography, Affinity; D002918:Chymotrypsin / Q000378:metabolism; D003429:Cross Reactions; D000939:Epitopes / Q000032:analysis; D005895:Gizzard, Avian / Q000032:analysis*; D007163:Immunosorbent Techniques; D008970:Molecular Weight; D010446:Peptide Fragments / Q000032:analysis; D013381:Subtilisins / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism
D000911:Antibodies, Monoclonal; D002148:Calmodulin-Binding Proteins; D000939:Epitopes; D010446:Peptide Fragments; D013381:Subtilisins; D002918:Chymotrypsin; D014357:Trypsin