Effect of substrate on Ca2(+)-concentration required for activity of the Ca2(+)-dependent proteinases, mu- and m-calpain.
The Ca2+ concentrations required for half-maximal activity of mu- and m-calpain purified from bovine skeletal muscle were tested using four different protein substrates and three different synthetic peptide substrates. Hammersten casein, the commonly used substrate for measuring mu- and m-calpain activity, required 2.5 microM Ca2+ for half-maximal activity of mu-calpain and 290 microM Ca2+ for half-maximal activity of m-calpain. When Hammersten casein was dialyzed against 8 M urea and 10 mM EDTA to remove all endogenous Ca2+, it required 1.9 and 290 microM Ca2+ for half-maximal activity of mu- and m-calpain, respectively. Rabbit skeletal muscle myofibrils and rabbit skeletal muscle troponin required 65 microM and 24 microM Ca2+ for half-maximal activity of mu-calpain and 380 microM and 580 microM Ca2+ for half-maximal activity of m-calpain, respectively. The three synthetic substrates tested, Suc-Leu-Tyr-MCA, Boc-Leu-Thr-Arg-MCA, and Suc-Leu-Leu-Val-Tyr-MCA, required 1.6 microM to 3.7 microM Ca2+ for half-maximal activity of mu-calpain and 200 to 560 microM Ca2+ for half-maximal activity of m-calpain.
Barrett
M J
MJ
Department of Animal Sciences, University of Arizona, Tucson 85721.
Goll
D E
DE
Thompson
V F
VF
D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002154:Calpain / Q000378:metabolism*; D002364:Caseins / Q000378:metabolism / Q000494:pharmacology*; D002417:Cattle; D007700:Kinetics; D008969:Molecular Sequence Data; D009210:Myofibrils / Q000378:metabolism; D009994:Osmolar Concentration; D010455:Peptides / Q000378:metabolism / Q000494:pharmacology; D013379:Substrate Specificity; D014336:Troponin / Q000378:metabolism / Q000494:pharmacology
D002364:Caseins; D010455:Peptides; D014336:Troponin; D002154:Calpain; D002118:Calcium