OXIDATION INHIBITS SUBSTRATE PROTEOLYSIS BY CALPAIN-I BUT NOT AUTOLYSIS

In this study, the effects of oxidation on calpain I autolysis and cal pain-mediated proteolysis were examined. Calpain I was incubated with increasing concentrations of free calcium in the presence or absence o f oxidant, and autolytic conversion of both the 80- and 30-kDa subunit s was measured by immunoblotting utilizing monoclonal antibodies which recognize both autolyzed and non-autolyzed forms of each subunit, res pectively. Autolytic conversion of the 80-kDa subunit of calpain I was not detected until free calcium concentration was greater than 40 mu M, whereas autolysis of the 30-kDa subunit did not occur until the fre e calcium concentration was greater than 100 mu M. In addition, autoly tic conversion of either the 80- or 30-kDa subunit was not inhibited b y the presence of oxidant. Calpain I activity was measured using the f luorescent peptide 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 belo w that required for autolysis. Calpain I activity was strongly inhibit ed by oxidant at all calcium concentrations studied, suggesting that p roteolytic activity of both the non-autolyzed 80-kDa and autolyzed 76- kDa forms was susceptible to oxidation. Interestingly, whereas oxidati on did not inhibit autolytic conversion, the presence of high substrat e concentrations did result in a significant reduction of autolysis wi thout altering calpain proteolytic activity. Calpain I activity that h ad been inhibited by the presence of oxidant was recovered immediately by addition of the reducing agent dithiothreitol.