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Ionic strength-induced inactivation of mu-calpain in postmortem muscle
1Roman L. Hruska U.S. Meat Animal Research Center, ARS, USDA, Clay Center, NE 68933-0166, USA.
Abstract:
The present study was conducted to study the stability of autolyzed mu-calpain activity and determine whether measurement of mu-calpain activity after anion exchange chromatography accurately reflects its activity in postmortem muscle. Ionic strength and pH affected the stability of partially autolyzed mu-calpain. Complete loss of activity was observed as a result of binding of autolyzed mu-calpain to DEAE-Sephacel when the large subunit of mu-calpain was autolyzed from 80 to 76 kDa. Therefore, determination of mu-calpain by standard anion exchange chromatography may underestimate mu-calpain activity in postmortem muscle. The activity of autolyzed mu-calpain was stabilized by inclusion of glycerol in the buffers, and this permitted us to investigate whether the apparent loss of mu-calpain activity in postmortem muscle is an artifact of the methodology. Despite the inclusion of glycerol in the buffers, a decrease in mu-calpain activity was observed during postmortem storage of muscle, even though the autolyzed enzyme was readily detectable by Western blotting in muscle extracts and column eluates. This result indicates that instability of autolyzed mu-calpain is a major cause for the decline in mu-calpain activity in postmortem muscle.
Insights
Autolyzed mu-calpain activity is unstable and can be underestimated by standard anion exchange chromatography. Instability during postmortem storage, not methodology, causes this decline in muscle tissue.
Area of Science:
- Biochemistry
- Muscle Physiology
Background:
- Mu-calpain is a key enzyme in postmortem muscle.
- Its activity is crucial for meat tenderization.
- Understanding mu-calpain stability is vital for meat science.
Purpose of the Study:
- To assess the stability of autolyzed mu-calpain.
- To determine if anion exchange chromatography accurately measures its activity in postmortem muscle.
- To investigate factors affecting autolyzed mu-calpain stability.
Main Methods:
- Investigated the effect of ionic strength and pH on autolyzed mu-calpain.
- Utilized anion exchange chromatography (DEAE-Sephacel).
- Employed glycerol to stabilize enzyme activity during experiments and Western blotting for detection.
Main Results:
- Autolyzed mu-calpain activity was sensitive to pH and ionic strength.
- Complete activity loss occurred upon binding to DEAE-Sephacel due to subunit degradation.
- Glycerol stabilized activity, but postmortem storage still decreased mu-calpain activity, confirmed by Western blot.
Conclusions:
- Standard anion exchange chromatography may underestimate mu-calpain activity in postmortem muscle.
- The decline in mu-calpain activity during postmortem storage is primarily due to enzyme instability.
- Autolyzed mu-calpain instability is a significant factor in postmortem muscle changes.