digitonin and calpastatin

To study nuclear transport of purified calpains in an in vitro system, A431 cells were permeabilized with digitonin, and fluorescein-labeled calpains were introduced under conditions known to facilitate energy-dependent nuclear transport of proteins. Fluorescein-mu-calpain was transported into nuclei in an ATP-dependent fashion. The calpain-specific inhibitor protein, calpastatin, could not block mu-calpain translocation. Fluorescein-calpastatin and fluorescein-m-calpain were poorly transported at best. In the presence of rat liver cytosolic factors, accumulation of nuclear mu-calpain was maximum at approximately 1 microM Ca2+, and no transport was observed at 0.3 microM Ca2+. Rat erythrocyte and HeLa cell extracts supported transport in the absence of Ca2+.

Topics: Amino Acid Sequence; Animals; Biological Transport; Calcium; Calcium-Binding Proteins; Calpain; Cattle; Cell Nucleus; Digitonin; Fluorescein; Fluoresceins; Humans; Molecular Sequence Data; Tumor Cells, Cultured

The integrin beta 4 subunit often undergoes proteolytic cleavage within its long cytoplasmic tail to yield a characteristic protein pattern of 205, 165, and 125 kDa. The results in this study suggest that beta 4 cleavage often occurs during or after cell lysis, where it was readily inhibitable by calcium chelators (EDTA, EGTA) and inhibitors of cysteine proteases (E64c, leupeptin). The cleavage of beta 4 is catalyzed by a calpain-like enzyme because (i) it requires calcium, (ii) it is mimicked by purified milli-calpain, and (iii) it is inhibited by several calpain inhibitors including the calpain-specific inhibitor calpastatin. Within intact cells, cleavage of beta 4 was cell type-specific and observed only when the cells were made permeable to calcium. Substantial cell viability was retained during beta 4 cleavage induced by ionomycin plus calcium, indicating that cleavage within intact cells was not necessarily a consequence of cell death. However, manipulations of cells including suspension, synchronization, and stimulation with serum, phorbol esters, or other agents all failed to induce cleavage, suggesting that if cleavage is physiologically relevant, it is not easily duplicated in vitro. Analysis of multiple cell types showed a wide variation in beta 4 sensitivity to proteolytic cleavage, suggesting that this process might be differentially regulated depending on the cellular environment.

Topics: Antigens, Surface; Artifacts; Calcium; Calcium-Binding Proteins; Calpain; Cell Adhesion; Cell Survival; Digitonin; Egtazic Acid; Endopeptidases; Humans; Integrin alpha6beta4; Integrin beta4; Integrins; Ionomycin; Peptide Fragments; Tumor Cells, Cultured