Subcellular localization of bovine heart calcium-dependent protease inhibitor

Four monoclonal antibodies for a calcium-dependent protease inhibitor protein were produced by fusing Sp2/0 myeloma cells with spleen cells from a Balb/C mouse immunized with purified bovine heart inhibitor. Each of the monoclonal antibodies was highly specific for the inhibitory protein as revealed by electro-blot analysis. The antibodies recognized different antigenic sites on CNBr peptides prepared from the purified inhibitor protein. Immunofluorescent microscopy of sections from bovine heart ventricles treated with each of the antibodies demonstrated the same fluorescent pattern. Fluorescence was observed at or near the sarcolemma of the myocytes, and along the Z-discs of relaxed myofibrils within the myocytes. Contracted myofibrils did not appear to bind antibody. Immunostaining of glycerinated relaxed cardiac myofibrils revealed staining at the Z-discs. One of the antibodies could also stain the Z-disc region of bovine skeletal muscle myofibrils.