Pressure effects on proteolysis catalysed by calpain

The effects of pressure on mu and m-calpain stability and specific activity have been examined. Activity and stability of these neutral calcium-depend ent heterodimeric proteinases were studied using an in-house built bioreact or allowing on-lint spectrophotometric monitoring with retention of pressur e. Both isozymes were founded to be rather bare-sensitive with t(1/2) at 15 00 bar of 6 min and 11 min for mu and m-calpain respectively. Activity meas urements under pressure showed a biphasic behavior for both proteinases wit h a slight activation for pressure up to 500 bar and 750 bar for m and mu-c alpain respectively. Activation volume changes indicated that the proteolyt ic reaction was alternatively favored and disfavored by pressure due to cat alytic 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 i ndicating that pressure inhibition of proteolytic activity could also be du e to Ca2+-binding decrease under pressure. Implication of these results wit h catalytic mechanism of these heterodimeric proteinases is also discussed.