EFFECT OF MONOCLONAL-ANTIBODIES SPECIFIC FOR THE 28-KDA SUBUNIT ON CATALYTIC PROPERTIES OF THE CALPAINS

Nine monoclonal antibodies (mAbs) specific for the 28-kDa subunit comm on to mu- and m-calpains have been assayed for their effects on mu- an d m-calpains. All nine react with the COOH-terminal part (domain VI) o f the 28-kDa subunit, and all nine affect the Ca2+ concentration requi red for autolysis of m-calpain, but have little effect on the Ca2+ con centration required for autolysis of mu-calpain. None of the nine affe ct the specific proteolytic activity of mu- or m-calpain. Two of the m Abs, 5B9 and 5B3, were selected for further study. mAb 5B9 decreased t he Ca2+ concentration required for autolysis to one-fifth of that requ ired in its absence; sequencing of chymotryptic fragments showed that the epitope for mAb 5B9 is between amino acid residues 92 and 104 of t he 28-kDa subunit. mAb 5B3 increased the Ca2+ concentration required f or autolysis; the epitope for mAb 5B3 is located between amino acid re sidues 148 and 178 of the 28-kDa subunit, which is the region that con tains the first EF-hand Ca2+-binding sequence in this subunit. Althoug h it increases the Ca2+ concentration required for autolysis, mAb 5B3 has no effect on the Ca2+ concentration required for proteolytic activ ity of m-calpain, and unautolyzed m-calpain is not a proenzyme. That a ll nine mAbs react with domain VI and not with the NH2-terminal domain V of the 28-kDa subunit suggests that domain VI (and not domain V) is involved in autolysis, contrary to the view that phosphatidylinositol lowers the Ca2+ concentration required for autolysis by binding to do main V.