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-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 mu 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 mu-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.
Bessiere
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P
Laboratoire de Biochimie et Technologie des Aliments, UA-429 INRA, Institut des Sciences et Techniques des Aliments de Bordeaux, Université Bordeaux I, Talence, France.
Bancel
F
F
Cottin
P
P
Ducastaing
A
A
D000818:Animals; D019149:Bioreactors; D002118:Calcium / Q000378:metabolism; D002154:Calpain / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D007700:Kinetics; D018482:Muscle, Skeletal / Q000378:metabolism; D011312:Pressure; D011817:Rabbits; D013997:Time Factors
D002154:Calpain; D002118:Calcium