Pressure effects on proteolysis catalysed by calpain

The effects of pressure on μ and m-calpain stability and specific activity have been examined. Activity and stability of these neutral calcium-dependent heterodimeric proteinases were studied using an in-house built bioreactor allowing on-line spectrophotometric monitoring with retention of pressure. Both isozymes were founded to be rather baro-sensitive with t1/2 at 1500 bar of 6 min and 11 min for μ and m-calpain respectively. Activity measurements under pressure showed a biphasic behavior for both proteinases with a slight activation for pressure up to 500 bar and 750 bar for m and μ-calpain respectively. Activation volume changes indicated that the proteolytic reaction was alternatively favored and disfavored by pressure due to catalytic step activation associated with enzyme-substrate binding step being continuously inhibited by pressure. Furthermore, autoproteolysis of calpain, a calcium dependent phenomenon was inhibited by application of pressure indicating that pressure inhibition of proteolytic activity could also be due to Ca2+-binding decrease under pressure. Implication of these results with catalytic mechanism of these heterodimeric proteinases is also discussed.