Ionic strength-induced inactivation of mu-calpain in postmortem muscle.
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.
Geesink
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Roman L. Hruska U.S. Meat Animal Research Center, ARS, USDA, Clay Center, NE 68933-0166, USA.
Koohmaraie
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D000818:Animals; D002135:Calcium-Binding Proteins / Q000378:metabolism; D002154:Calpain / Q000378:metabolism*; D002852:Chromatography, Ion Exchange / Q000662:veterinary; D015853:Cysteine Proteinase Inhibitors / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel / Q000662:veterinary; D006863:Hydrogen-Ion Concentration; D009132:Muscles / Q000201:enzymology*; D009994:Osmolar Concentration; D011180:Postmortem Changes; D013552:Swine
D002135:Calcium-Binding Proteins; D015853:Cysteine Proteinase Inhibitors; C033668:calpastatin; D002154:Calpain