Interaction between catalytically inactive calpain and calpastatin. Evidence for its occurrence in stimulated cells.
Conformational changes in the calpain molecule following interaction with natural ligands can be monitored by the binding of a specific monoclonal antibody directed against the catalytic domain of the protease. None of these conformational states showed catalytic activity and probably represent intermediate forms preceding the active enzyme state. In its native inactive conformation, calpain shows very low affinity for this monoclonal antibody, whereas, on binding to the ligands Ca(2+), substrate or calpastatin, the affinity increases up to 10-fold, with calpastatin being the most effective. This methodology was also used to show that calpain undergoes similar conformational changes in intact cells exposed to stimuli that induce either a rise in intracellular [Ca(2+)] or extensive diffusion of calpastatin into the cytosol without affecting Ca(2+) homeostasis. The fact that the changes in the calpain state are also observed under the latter conditions indicates that calpastatin availability in the cytosol is the triggering event for calpain-calpastatin interaction, which is presumably involved in the control of the extent of calpain activation through translocation to specific sites of action.
Averna
Monica
M
Department of Experimental Medicine (DIMES), Section of Biochemistry and Centre of Excellence for Biomedical Research (CEBR), University of Genova, Italy.
Stifanese
Roberto
R
De Tullio
Roberta
R
Defranchi
Enrico
E
Salamino
Franca
F
Melloni
Edon
E
Pontremoli
Sandro
S
D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism*; D001665:Binding Sites; D001921:Brain / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D002154:Calpain / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002384:Catalysis; D015853:Cysteine Proteinase Inhibitors / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D004912:Erythrocytes / Q000378:metabolism; D006801:Humans; D019169:Jurkat Cells; D008024:Ligands; D009504:Neutrophils / Q000378:metabolism; D011487:Protein Conformation; D051381:Rats
D000911:Antibodies, Monoclonal; D002135:Calcium-Binding Proteins; D015853:Cysteine Proteinase Inhibitors; D008024:Ligands; C033668:calpastatin; D002154:Calpain; D002118:Calcium