Effect of monoclonal antibodies specific for the 28-kDa subunit on catalytic properties of the calpains.

Abstract

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.

• Authors: J Cong, V F Thompson, D E Goll
• Journal: The Journal of biological chemistry (J Biol Chem) Vol. 268 Issue 34 Pg. 25740-7 (Dec 05 1993) ISSN: 0021-9258 [Print] United States
• PMID: 7503986 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't, Research Support, U.S. Gov't, Non-P.H.S., Research Support, U.S. Gov't, P.H.S.)

Chemical References

• Antibodies, Monoclonal
• Epitopes
• Macromolecular Substances
• Calpain
• Calcium

Topics

• Amino Acid Sequence
• Animals
• Antibodies, Monoclonal (pharmacology)
• Binding Sites
• Blotting, Western
• Calcium (pharmacology)
• Calpain (immunology, isolation & purification, metabolism)
• Electrophoresis, Polyacrylamide Gel
• Enzyme-Linked Immunosorbent Assay
• Epitopes (analysis)
• Female
• Humans
• Kinetics
• Macromolecular Substances
• Mice
• Mice, Inbred BALB C (immunology)
• Molecular Sequence Data