SUBSTRUCTURE OF NEBULIN FILAMENTS - LOCALIZATION AND CHARACTERIZATIONOF SUBFRAGMENTS PRODUCED BY 0.1 MM CACL2

The locations of five kinds of nebulin subfragments in chicken myofibr ils were studied by indirect immunofluorescence microscopy and immunoe lectron microscopy. The subfragments were produced by treatment of the myofibrils with a solution containing 0.1 mM CaCl2. Antinebulin-subfr agment antibodies displayed five stripes from the Z-disk to the distal end of thin filaments in each half sarcomere. Anti-40-kDa subfragment antibodies provided a wide stripe near the Z-disk. Anti-33-kDa subfra gment antibodies displayed three stripes in the I-band. Anti-23-kDa su bfragment antibodies displayed three stripes, whose positions could no t be distinguished from those of three stripes provided by anti-33-kDa subfragment antibodies. Anti-180-kDa subfragment antibodies provided fluorescence at the A-I junction region. Anti-200-kDa subfragment anti bodies displayed a single stripe at the distal end region of thin fila ments. The location of these stripes corresponded well to that of the mother protein, nebulin. On the basis of these results, we propose a m odel for the substructure of chicken nebulin filaments. All the nebuli n subfragments possessed the property of binding to F-actin, indicatin g that nebulin filaments bind to thin filaments along their entire len gth in situ. There is a possibility that nebulin filaments are anchore d at the Z-disk through interaction with some other Z-disk constituent s than alpha-actinin, because the binding site for alpha-actinin exist s on the distal end region of nebulin filaments.