抄録
We examined the function of β-actinin as a pointed end capping protein of thin filaments in skeletal muscle. An improvement in preparing β-actinin yielded purified β-actinin which retained its activity for more than a week. Two-dimensional gel electrophoresis showed that the two subunits, βI and βII, of β-actinin are, respectively, split into two to three components (isoforms) with different isoelectric points. Polyclonal antibody was raised by injecting such purified and undenatured chicken breast muscle β-actinin composed of several components into a rabbit. Immuno-gold labeling examination with electron microscopy of an F-actin-β-actinin complex decorated with HMM showed that 85% of bound gold particles was on the pointed end of actin filaments, while the remaining 15% was on the barbed end. This suggests that in β-actinin preparation pointed end and barbed end capping proteins inevitably coexist. Immunofluorescence and immunoelectron microscopy directly showed that β-actinin is located at the pointed end of thin filaments in myofibrils; it was also suggested that a capping protein having common antigenic determinants to β-actinin is located at Z-line. Thus, the physiological function of β-actinin as a pointed end capping protein was examined as follows: When β-actinin was dissociated from the pointed end of thin filaments in an I-Z-I brush by using a high salt solution, thin filaments could be disassembled at the pointed ends at concentrations of exogenous actin lower than a critical value. At a physiological ionic strength, these salt-washed thin filaments gradually shortened at a constant rate of about 45 nm/h. Both the association and dissociation of monomeric actin at the pointed end were suppressed by the rebinding of exogenous β-actinin. The main physiological role of β-actinin is therefore to stabilize thin filaments in the sarcomere by preventing addition and removal of actin monomers at the pointed filament end.
