EVIDENCE FOR TISSUE-ASSOCIATED ALPHA-(2)-MACROGLOBULIN IN MOUSE SKELETAL-MUSCLE

alpha(2)-Macroglobulin (alpha(2)M), a major serum protease inhibitor, was localized in mouse skeletal muscle by immunoperoxidase histochemis try. In all muscles examined (mm. soleus, plantaris, and extensor digi torum longus) specific immunoreactivity occurred diffusely in extracel lular structures (periendomysium, blood vessel wall) as well as inside about a half of the muscle fibers. This localization pattern did not change substantially by extensively perfusing deeply anesthetized mice with phosphate buffered saline (PBS) to remove serum alpha(2)M In rel ease experiments on fresh (nonfixed) cryostat sections, specific immun oreactivity persisted after an extensive prewash with PBS (up to 5-6 h ), but a new specific staining appeared inside those fibers that were originally negative. Western blotting experiments were negative on the soluble fraction of muscle homogenate, thus confirming that the perfu sion procedure was effective in removing serum alpha(2)M. By contrast, three specific bands (185, 165, and 35 kDa) appeared in detergent-sol ubilized extracts (0.3% Triton X-100), indicating the occurrence of ti ssue-associated alpha(2)M. Confocal immunofluorescence microscopy reve aled that the intracellular specific staining was associated to a long itudinal network, probably corresponding to the sarcoplasmic reticulum . A multifunctional role of alpha(2)M in skeletal muscle was hypothesi zed.