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 histochemistry. In all muscles examined (mm. soleus, plantaris, and extensor digitorum longus) specific immunoreactivity occurred diffusely in extracellular 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 release experiments on fresh (nonfixed) cryostat sections, specific immunoreactivity 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 perfusion procedure was effective in removing serum alpha(2)M. By contrast, three specific bands (185, 165, and 35 kDa) appeared in detergent-solubilized extracts (0.3% Triton X-100), indicating the occurrence of tissue-associated alpha(2)M. Confocal immunofluorescence microscopy revealed that the intracellular specific staining was associated to a longitudinal network, probably corresponding to the sarcoplasmic reticulum. A multifunctional role of alpha(2)M in skeletal muscle was hypothesized.
Fumagalli
L
L
Departement of Cardiovascular Sciences, University "La Sapienza," Rome, Italy.
Businaro
R
R
Nori
S L
SL
Toesca
A
A
Emmons
C
C
De Renzis
G
G
D000818:Animals; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D006614:Hindlimb; D007124:Immunoenzyme Techniques; D051379:Mice; D008810:Mice, Inbred C57BL; D018613:Microscopy, Confocal; D018485:Muscle Fibers, Skeletal / Q000166:cytology; D018482:Muscle, Skeletal / Q000098:blood supply / Q000737:chemistry / Q000166:cytology*; D009928:Organ Specificity; D010477:Perfusion; D000511:alpha-Macroglobulins / Q000032:analysis*
D000511:alpha-Macroglobulins