COMPARISON OF THE AUTOLYZED AND UNAUTOLYZED FORMS OF MU-AND M-CALPAIN FROM BOVINE SKELETAL-MUSCLE

Bovine skeletal muscle-mu- and m-calpain autolyze when incubated with Ca2+. During the first 30 to 300 s, autolysis: (1) has little effect on the specific proteolytic activity of either-mu- or m-calpain when assayed at 5 mM Ca2+; and (2) produces two new proteolytically active forms of calpain in addition to the original-mu- and m-calpain. The four proteolytically active forms of calpain are: (1) autolyzed-mu-calpain, having polypeptide subunits of 76 and 18 kDa and requiring 0.60-mu-M Ca2+ for half-maximal activity; (2) mu-calpain with 80- and 28-kDa subunits and requiring 7.1-mu-M Ca2+ for half-maximal activity; (3) autolyzed m-calpain with 78- and 18-kDa subunits and requiring 180-mu-M Ca2+ for half-maximal activity; and (4) m-calpain with 80- and 28-kDa subunits and requiring 1000-mu-M Ca2+ for half-maximal activity. All four forms of the calpains have similar pH optima (7.4 to 7.6) and almost identical circular dichroism spectra in the far ultraviolet (all four have little secondary structure with 26-30% alpha-helix and less than 10% beta-sheet structure). Autolyzed-mu- and unautolyzed-mu-calpain are fully activated proteolytically by Mn2+ with activity starting at 125-mu-M Mn2+. Autolyzed m-calpain is also activated by Mn2+ up to 80% of the maximum proteolytic activity obtained with Ca2+; Mn2+ activation begins at 320-mu-M Mn2+. Unautolyzed m-calpain has only 6 to 8% as much activity in the presence of Mn2+ as it does in the presence of Ca2+. Autolysis increases the axial ratios of the calpains from 3.5 to 4.6 for mu-calpain and from 3.7 to 5.0 for m-calpain (assuming 20% hydration). The estimated length of the calpain molecules increases by 13% upon autolysis from 73 to 84 angstrom for mu-calpain and from 76 to 90 angstrom for m-calpain (assuming 20% hydration). The autolyzed calpains elute after their unautolyzed counterparts off a DEAE-ion exchange column. Because autolyzed forms of the calpains are not found in DEAE elution profiles of cell extracts, bovine skeletal muscle cells must contain very little (less than 5% of total calpain) or none of the autolyzed form of the calpains.