Effect of pH, temperature, and inhibitors on autolysis and catalytic activity of bovine skeletal muscle mu-calpain.
To improve our understanding of the regulation of calpain activity in situ during postmortem storage, the effects of pH, temperature, and inhibitors on the autolysis and subsequent proteolytic activity of mu-calpain were studied. Calpains (mu- and m-calpain) and calpastatin were purified from bovine skeletal muscle. All autolysis experiments were conducted in the absence of substrate at different pH (7.0, 6.2, and 5.8) and temperatures (25 and 5 degrees C). Autolysis of mu-calpain generated polypeptides with estimated masses of 61, 55, 40, 27, 23, and 18 kDa. The rate of autolysis was significantly increased with decreasing pH. The rate of degradation of the 80-kDa subunit was significantly decreased with decreasing temperature. However, degradation of the 30-kDa subunit was not affected by decreasing temperature. By conducting autolysis experiments at 5 degrees C and immunoblotting of autolytic fragments with anti-80 kDa, it was demonstrated that with the exception of 18 kDa, which originates from 30 kDa, all other fragments probably originate from degradation of the 80-kDa subunit. Calpastatin, leupeptin, and E-64 did not inhibit the initial step of autolysis, but they did inhibit further breakdown of these fragments. However, zinc, which also inhibits the proteolytic activity of calpain, only reduced the rate of autolysis, but did not inhibit it. The possible significance of these results in terms of the regulation of calpain in postmortem muscle is discussed.
Koohmaraie
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Roman L. Hruska U.S. Meat Animal Research Center, ARS, USDA, Clay Center, NE 68933-0166.
D000818:Animals; D001329:Autolysis; D002135:Calcium-Binding Proteins / Q000494:pharmacology; D002154:Calpain / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002417:Cattle / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D009132:Muscles / Q000201:enzymology*; D011180:Postmortem Changes; D013696:Temperature
D002135:Calcium-Binding Proteins; C033668:calpastatin; D002154:Calpain