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Oxidation Inhibits Substrate Proteolysis by Calpain I but Not Autolysis

Oxidation Inhibits Substrate Proteolysis by Calpain I but Not Autolysis

R P Guttmann, J S Elce, P D Bell, J C Isbell, G V Johnson

J Biol Chem. 1997 Jan 17;272(3):2005-12.

PMID: 8999893

Abstract:

In this study, the effects of oxidation on calpain I autolysis and calpain-mediated proteolysis were examined. Calpain I was incubated with increasing concentrations of free calcium in the presence or absence of oxidant, and autolytic conversion of both the 80- and 30-kDa subunits was measured by immunoblotting utilizing monoclonal antibodies which recognize both autolyzed and non-autolyzed forms of each subunit, respectively. Autolytic conversion of the 80-kDa subunit of calpain I was not detected until free calcium concentration was greater than 40 microM, whereas autolysis of the 30-kDa subunit did not occur until the free calcium concentration was greater than 100 microM. In addition, autolytic conversion of either the 80- or 30-kDa subunit was not inhibited by the presence of oxidant. Calpain I activity was measured using the fluorescent peptide N-succinyl-L-leucyl-L-leucyl-L-valyl-L-tyrosine-7-amido-4- methylcoumarin or the microtubule-associated protein tau as substrate. Calpain I was found to have proteolytic activity at free calcium concentrations below that required for autolysis. Calpain I activity was strongly inhibited by oxidant at all calcium concentrations studied, suggesting that proteolytic activity of both the non-autolyzed 80-kDa and autolyzed 76-kDa forms was susceptible to oxidation. Interestingly, whereas oxidation did not inhibit autolytic conversion, the presence of high substrate concentrations did result in a significant reduction of autolysis without altering calpain proteolytic activity. Calpain I activity that had been inhibited by the presence of oxidant was recovered immediately by addition of the reducing agent dithiothreitol.

Chemicals Related in the Paper:

Catalog Number	Product Name	Structure	CAS Number	Price
AP94099577	L-Tyrosine 7-amido-4-methylcoumarin		94099-57-7	Price

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