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 common to mu- and m-calpains have been assayed for their effects on mu- and m-calpains. All nine react with the COOH-terminal part (domain VI) of the 28-kDa subunit, and all nine affect the Ca2+ concentration required for autolysis of m-calpain, but have little effect on the Ca2+ concentration required for autolysis of mu-calpain. None of the nine affect the specific proteolytic activity of mu- or m-calpain. Two of the mAbs, 5B9 and 5B3, were selected for further study. mAb 5B9 decreased the Ca2+ concentration required for autolysis to one-fifth of that required in its absence; sequencing of chymotryptic fragments showed that the epitope for mAb 5B9 is between amino acid residues 92 and 104 of the 28-kDa subunit. mAb 5B3 increased the Ca2+ concentration required for autolysis; the epitope for mAb 5B3 is located between amino acid residues 148 and 178 of the 28-kDa subunit, which is the region that contains the first EF-hand Ca(2+)-binding sequence in this subunit. Although it increases the Ca2+ concentration required for autolysis, mAb 5B3 has no effect on the Ca2+ concentration required for proteolytic activity of m-calpain, and unautolyzed m-calpain is not a proenzyme. That all 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 domain V.
Cong
J
J
Muscle Biology Group, University of Arizona, Tucson 85721.
Thompson
V F
VF
Goll
D E
DE
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology*; D001665:Binding Sites; D015153:Blotting, Western; D002118:Calcium / Q000494:pharmacology; D002154:Calpain / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000032:analysis; D005260:Female; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D051379:Mice; D008807:Mice, Inbred BALB C / Q000276:immunology; D008969:Molecular Sequence Data
D000911:Antibodies, Monoclonal; D000939:Epitopes; D046911:Macromolecular Substances; D002154:Calpain; D002118:Calcium