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.