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.
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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.)
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Chemical References |
- Antibodies, Monoclonal
- Epitopes
- Macromolecular Substances
- Calpain
- Calcium
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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
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