Ionic strength-induced inactivation of mu-calpain in postmortem muscle

The present study was conducted to study the stability of autolyzed mu-calp ain activity and determine whether measurement of mu-calpain activity after anion exchange chromatography accurately reflects its activity in postmort em muscle. Ionic strength and pH affected the stability of partially autoly zed mu-calpain. Complete loss of activity was observed as a result of bindi ng of autolyzed CL-calpain to DEAE-Sephacel when the large subunit of mu-ca lpain was autolyzed from 80 to 76 kDa. Therefore, determination of mu-calpa in by standard anion exchange chromatography may underestimate mu-calpain a ctivity in postmortem muscle. The activity of autolyzed mu-calpain was stab ilized by inclusion of glycerol in the buffers, and this permitted us to in vestigate whether the apparent loss of mu-calpain activity in postmortem mu scle is an artifact of the methodology. Despite the inclusion of glycerol i n the buffers, a decrease in mu-calpain activity was observed during postmo rtem storage of muscle, even though the autolyzed enzyme was readily detect able by Western blotting in muscle extracts and column eluates. This result indicates that instability of autolyzed mu-calpain is a major cause for th e decline in mu-calpain activity in postmortem muscle.