Immunochemical analysis of alpha-actinin of nemaline myopathy after two-dimensional electrophoresis.

We analyzed alpha-actinin from human skeletal muscle by immunoblotting after two-dimensional electrophoresis. A monoclonal antibody, S alpha 5-17, was established after immunization in Balb/c mouse with crude alpha-actinin fraction from human soleus muscle. Western blotting and indirect immunofluorescence microscopy revealed that the antibody reacted selectively with alpha-actinin from human skeletal muscle and stained in a manner equivalent to that of type 1, 2A, 2B and 2C myofibers and cardiac atrial and ventricular muscles. No reactivity was observed in the arterial smooth muscle layer or in the central and peripheral nervous systems. The antibody exhibited 2 spots with different isoelectric points in a range more basic than that of actin upon immunoblotting after two-dimensional gel electrophoresis, suggesting the presence of 2 variants of alpha-actinin in human skeletal muscle. Analysis of type 2B-deficient muscle with nemaline myopathy or central core disease revealed that type 2B myofibers contained the basic variant, while type 1 and 2A myofibers contained only the acidic variant. Immunoblots performed after two-dimensional gel electrophoresis of muscles with nemaline myopathy revealed alpha-actinin variants indistinguishable from those of control muscles.