Effect of mu-calpain on m-calpain

The free Ca2+ concentrations required for half-maximal proteolytic activity of m-calpain are in the range of 400-800 mu M and are much higher than the 50-500 nM free Ca2+ concentrations that exist in living cells. Consequentl y, a number of studies have attempted to find mechanisms that would lower t he Ca2+ concentration required for proteolytic activity of m-calpain. Altho ugh autolysis lowers the Ca2+ concentration required for proteolytic activi ty of m-calpain, 90-400 mu M Ca2+ is required for a half-maximal rate of au tolysis of m-calpain, even in the presence of phospholipid. It has been sug gested that mu-calpain, which has a lower Ca2+ requirement than m-calpain, might proteolyze m-calpain and reduce its Ca2+ requirement to a level that would allow it to be active at physiological Ca2+ concentrations. We have i ncubated m-calpain with mu-calpain for 60 min at a ratio of 1:50 mu-calpain : m-calpain, in the presence of 50 mu M free Ca2+; this Ca2+ concentration is high enough for more than halfmaximal activity of mu-calpain, but does n ot activate m-calpain, Under these conditions, mu-calpain caused no detecta ble proteolytic degradation of the m-calpain polypeptide and did not change the Ca2+ concentration required for proteolytic activity of m-calpain, mu- Calpain also did not degrade the m-calpain polypeptide at 1000 mu M Ca2+, w hich is a Ca2+ concentration high enough to completely activate m-calpain. It seems unlikely that mu-calpain could act as an "activator" of m-calpain in living cells. Because m-calpain rapidly degrades itself (autolyzes) at 1 000 mu M Ca2+ and because the subsite specificities of mu- and m-calpain ar e very similar if not identical, failure of mu-calpain to rapidly degrade m -calpain at 1000 mu M Ca2+ suggests a unique role of autolysis in calpain f unction. (C) 2000 Academic Press.