calpastatin and Hemolysis

The effects of low intensity, low frequency magnetic fields (MFs) on catalytic activity of the calcium dependent protease calpain was determined following the enzyme activation both in "in vitro" and "in vivo" conditions. We have observed that a 0.3 mT MF induces a significant increase in the requirement of the protease for this metal ion. This change is detectable at low [Ca(2+)] and disappears when the level of Ca(2+) is raised to saturating amounts. The observed effects are not due to transient MF(-) induced conformational changes occurring in calpain, but to direct effects of the MF on Ca(2+) ions, which become less available for the binding sites present in calpain. Altogether, these results indicate that exposure to low intensity, low frequency MFs alters the intracellular Ca(2+) "availability," thereby modifying the related cell response.

Topics: Animals; Anion Exchange Protein 1, Erythrocyte; Calcium; Calcium-Binding Proteins; Calpain; Electromagnetic Fields; Enzyme Activation; Erythrocytes; Hemolysis; Humans; Rats

A highly sensitive sandwich enzyme immunoassay for a Ca2+-dependent cysteine proteinase (calpain I) and its specific endogenous inhibitor protein (calpastatin) was developed. The calpain I and calpastatin used as immunogens were purified from human erythrocytes. Anti-calpastatin antisera having sufficiently high titer were obtained only when the immunogen was purified by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The assay method was principally based on the report by M. Imagawa et al. (1982, J. Appl. Biochem. 4, 41-57), using a specific antibody-coated polystyrene ball and horseradish peroxidase-conjugated Fab' fragment of the antibody. The sensitivity was 0.1 ng of calpain I or calpastatin per assay tube. Starting with 50 microliter of the hemolysate from human erythrocytes, the method permitted direct and simultaneous determination of calpain I and calpastatin, without prior separation of these two enzymatically counteracting components by chromatography. The present method as applied to the erythrocytes from 14 healthy adults gave 120-170 micrograms for calpain I and 164-211 micrograms for calpastatin per gram of hemoglobin, respectively.

Topics: Calcium-Binding Proteins; Calpain; Endopeptidases; Erythrocytes; Hemolysis; Humans; Immunoenzyme Techniques; Methods; Protease Inhibitors