Oxidation inhibits substrate proteolysis by calpain I but not autolysis.
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.
Guttmann
R P
RP
Department of Psychiatry, University of Alabama at Birmingham, Birmingham, Alabama 35294-0017, USA.
Elce
J S
JS
Bell
P D
PD
Isbell
J C
JC
Johnson
G V
GV
D002118:Calcium / Q000378:metabolism; D002154:Calpain / Q000378:metabolism*; D018076:DNA, Complementary; D004229:Dithiothreitol / Q000737:chemistry; D006801:Humans; D006868:Hydrolysis; D007202:Indicators and Reagents; D010084:Oxidation-Reduction; D013379:Substrate Specificity
D018076:DNA, Complementary; D007202:Indicators and Reagents; D002154:Calpain; D002118:Calcium; D004229:Dithiothreitol