title: Isolation and characterization of a conserved actin-binding domain from rat hepatic actinogelin, rat skeletal muscle, and chicken gizzard alpha-actinins.
abstract: A common protease-resistant fragment (Mr = 27,000) was generated from purified rat hepatic actinogelin, and rat skeletal muscle and chicken gizzard alpha-actinins by limited proteolysis using thermolysin. A monoclonal antibody (A-1) which was raised against the rat hepatic actinogelin and has a cross-reactivity with rat skeletal muscle and chicken gizzard alpha-actinins was found to bind to all of the 27-kDa fragments selectively. Furthermore, one-dimensional peptide maps of the 27-kDa fragments showed a close similarity indicating the presence of some conservation in primary structure of the fragments. The 27-kDa fragments were purified to homogeneity by the same procedure using ammonium sulfate fractionation and hydrophobic chromatography. As the fragments were easily separated from other peptides during purification, they might be present as an independent structural domain. Purified 27-kDa fragments were found to bind to F-actin in a Ca2+-insensitive manner. The fragments failed to affect the low-shear viscosity of F-actin up to a molar ratio to actin monomer of 1:3.2, indicating that gelation activity of the parental molecules was lost and the fragments have only a single binding site on F-actin. Binding of the fragments to F-actin was almost completely inhibited by respective parental molecules, while binding of the whole molecules was blocked partly by their 27-kDa fragments. Thus, the interaction of the fragments with F-actin seemed to be specific, although apparent affinity was lower than the parental molecules. Considering these results, we concluded that the 27-kDa fragments are a conserved, monovalent, and Ca2+-insensitive actin-binding domain of the actinogelin and muscle alpha-actinins.
mesh_terms: D000185:Actinin / Q000378:metabolism*; D000199:Actins / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002645:Chickens; D005895:Gizzard, Avian / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000378:metabolism*; D008840:Microfilament Proteins / Q000378:metabolism*; D009130:Muscle, Smooth / Q000378:metabolism*; D009132:Muscles / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism; D011485:Protein Binding; D051381:Rats; D013820:Thermolysin
chemical_list: D000199:Actins; D008840:Microfilament Proteins; D010446:Peptide Fragments; C029354:actinogelin; D000185:Actinin; D013820:Thermolysin