Detecting the active conformation of calpain with calpastatin-based reagents

Engineering Calpastatin to Develop a Sensor to Detect Active Calpain

PROBING THE ACTIVE SITE OF RHODANESE WITH DISULFIDE REAGENTS

A simple one-step procedure for the separation of calpain I, calpain II and calpastatin.

The soluble fraction from rabbit brain was adsorbed on a column of phenyl-Sepharose. By applying a linear gradient with decreasing salt concentration and increasing pH, it was possible to separate calpain I and calpain II from each other and from the endogenous inhibitor calpastatin. Both enzymes were capable of degrading endogenously labelled neuronal proteins, including slowly axonally transp...

probing the active site of rhodanese with disulfide reagents

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Caspase-mediated fragmentation of calpain inhibitor protein calpastatin during apoptosis.

Two cysteine protease families (caspase and calpain) participate in apoptosis. Here we report that the endogenous calpain inhibitor calpastatin is fragmented by caspase(s) to various extents during early apoptosis in two cell types. In anti-fas or staurosporine-treated Jurkat T-cells, the high-molecular-weight form (HMW) of calpastatin (apparent Mr 110 K) was extensively degraded to immunoreact...