A recent hypothesis suggests that proteolytic activity of the micromolar and millimolar Ca2+-requiring forms of the Ca2+-dependent
proteinases (mu- and
m-calpain, respectively) is regulated in vivo by their association with a
phosphatidylinositol-containing site on the plasma membrane followed by
autolysis of the
proteinases.
Phosphatidylinositol association lowers the Ca2+ concentration needed for
autolysis, and
autolysis, in turn, lowers the Ca2+ concentration needed for proteolytic activity. To test this hypothesis, we have compared the Ca2+ concentrations needed for
autolysis and for proteolytic activity of the calpains both in the presence and the absence of
phosphatidylinositol. Bovine skeletal muscle
mu-calpain required 40-50 microM Ca2+ for half-maximal rate of proteolysis of a
casein substrate, 140-150 microM Ca2+ for half-maximal
autolysis in the presence of 80 microM
phosphatidylinositol, and 190-210 microM Ca2+ for half-maximal
autolysis in the absence of
phosphatidylinositol. Consequently,
mu-calpain is an active
proteinase and does not require
autolysis for activation. Bovine skeletal muscle
m-calpain required 700-740 microM Ca2+ for half-maximal rate of proteolysis of a
casein substrate, 370-400 microM Ca2+ for half-maximal
autolysis in the presence of 80 microM
phosphatidylinositol, and 740-780 microM Ca2+ for half-maximal
autolysis in the absence of
phosphatidylinositol. These results are consistent with the idea that
m-calpain functions in its autolyzed form, but the results do not demonstrate that unautolyzed
m-calpain is inactive. 80 microM
phosphatidylinositol had no effect on the Ca2+ requirement of the autolyzed forms of either mu- or
m-calpain but lowered the specific activity of
mu-calpain to 20% of its activity in the absence of
phosphatidylinositol. Of the four forms of the calpains, unautolyzed
m-calpain, autolyzed
m-calpain, and unautolyzed
mu-calpain would not be proteolytically active at the free Ca2+ concentrations of 300-1200 nM present inside normal cells, and neither mu- nor
m-calpain would undergo
autolysis at these Ca2+ concentrations, even in the presence of
phosphatidylinositol. Cells must contain a mechanism other than or in addition to membrane association and
autolysis to activate the calpains.