Immunogold Electron-Microscopic Localization of Calpain I in Human Erythrocytes

Summary

With a view to understanding the function of calpain I (EC 3.4.22.17) in vivo, the localization of the enzyme was studied by immunoelectron microscopy in human erythrocytes. Thin sections of the cells embedded in Spurr’s resin were exposed to solutions of monoclonal anti-calpain I or control antibodies, biotin antimouse IgG antibodies, and streptavidin-gold. Most of the calpain I (93%) was found to be distributed throughout the cytoplasm, and only 7% of the gold particles were associated with the erythrocyte membrane. Erythrocytes were Ca²⁺-loaded by means of the calcium ionophore A23187, and the rise in intracellular [Ca²⁺] was demonstrated both by crenation of the cells, and by activation of calpain which was detected by immunoblotting. The proportions of cytosolic and membrane-bound gold labelling were, however not altered by Ca²⁺-loading. These results are not consistent with the hypothesis that activation of calpain requires membrane-binding.

Keywords

Calpain - Erythrocytes - Immunohistochemistry - Ca²⁺ - loading

• References

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