Localization and characterization of an intermediate filament-associated protein

A monoclonal antibody designated Mab G3.5 was used to test for the presence of reactive antigen in rat skeletal and cardiac muscle cells. Standard indirect labelling methods for immunofluorescence microscopy and immuno-gold localization for electron microscopy were applied to paraformaldehyde-fixed muscle tissues. In longitudinal sections, the reactive antigen was found to localize appropriately for Z-disks. In transverse sections of skeletal muscle, the antigen was apparent as a strongly fluorescent reticular network. Gold particles were observed by transmission electron microscopy in association with filamentous structures on either side of sarcomeres, and on either side of, but not adjacent to the Z-disk. This appearance is consistent with the exosarcomeric cytoskeleton and the known distribution of desmin. In competition binding experiments, neither antidesmin nor Mab G3.5 interfered with the binding of each other and co-localization was observed. Desmin (M(r) 52,000) differs in its relative molecular weight from the G3.5 antigen (M(r) 100,000), which was isolated and analysed by SDS-PAGE. The antigen was found to co-isolate with the cytoskeletal fraction from cell homogenates, and could be released from this fraction with the addition of reducing agents. Preliminary sequence analysis indicates that the G3.5 antigen may be an isoform of alpha-actinin which interconnects intermediate filaments and actin.