Effect of pH and ionic strength on bovine m-calpain and calpastatin activity.
The effects of bovine skeletal muscle m-calpain and calpastatin on the degradation of casein and isolated bovine myofibrils were characterized under various pH values (7.0, 6.2, 5.7) and ionic strengths (32 to 400 mM KCl) at 25 degrees C. Caseinolytic assays indicated that m-calpain activity increased with increasing pH (P < .01) but decreased with increasing ionic strength (P < .01). Regardless of the presence of m-calpain, SDS-PAGE of myofibrils showed increased solubilization of myofibrillar proteins as pH and ionic strength increased. However, only in the presence of m-calpain were changes normally observed during postmortem storage reproduced. Protein release attributed to m-calpain activity increased with pH, but the effects of elevated ionic strength on the ability of m-calpain to hydrolyze myofibrillar proteins were not evident from SDS-PAGE, except for the decreased troponin-T degradation by m-calpain at the higher ionic strengths. A pH x ionic strength interaction was observed for calpastatin activity determined by caseinolytic assays (P < .01). No changes in m-calpain inhibition were detected at pH 7.0 and 6.2 at different ionic strengths. However, at pH 5.7 the ability of calpastatin to inhibit m-calpain decreased with increasing ionic strength. No changes in m-calpain inhibition could be detected with SDS-PAGE. Based on these results, it can be concluded that although m-calpain and calpastatin activities decrease with increasing ionic strength, their activities in the presence of myofibrils were not affected by ionic strengths typically found in postmortem muscle.
Kendall
T L
TL
Department of Animal Science, University of Georgia, Athens 30602.
Koohmaraie
M
M
Arbona
J R
JR
Williams
S E
SE
Young
L L
LL
D000818:Animals; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D002154:Calpain / Q000378:metabolism*; D002364:Caseins / Q000378:metabolism; D002417:Cattle / Q000378:metabolism*; D046508:Culture Techniques; D006863:Hydrogen-Ion Concentration; D009124:Muscle Proteins / Q000378:metabolism; D009132:Muscles / Q000201:enzymology* / Q000378:metabolism; D009994:Osmolar Concentration
D002135:Calcium-Binding Proteins; D002364:Caseins; D009124:Muscle Proteins; C033668:calpastatin; D002154:Calpain