Calpastatin has two distinct sites for interaction with calpain--effect of calpastatin fragments on the binding of calpain to membranes.
Calpain, a ubiquitously distributed intracellular cysteine protease in animals, is thought to be activated at the cell membrane in the presence of micromolar levels of calcium ions. Calpastatin, the sole specific proteinaceous inhibitor for calpain, inhibits not only proteolytic activity, but also the binding of calpain to cell membranes. The inhibition of calpain binding to membranes by calpastatin fragments was analyzed. A calpastatin peptide containing the inhibitory sequence did not inhibit the binding of calpain to membranes. On the other hand, binding was prevented by a fragment without the activity to inhibit proteolytic activity. These data indicate that calpain binds to cell membranes through a site (regulatory site) other than the active site and that calpastatin inhibits the binding of calpain to cell membranes via a site (regulatory inhibition site) other than the inhibitory sequence. Calpain and calpastatin can undergo independent interactions: interaction between the catalytic site of calpain and the inhibitory sequence of calpastatin, and interaction between the regulatory site of calpain and the regulatory inhibition site of calpastatin. The interaction between the regulatory site of calpain and the regulatory inhibition site of calpastatin is essential for the regulation of calpain activity.
Department of Molecular Biology, Tokyo Metropolitan Institute of Medical Science, Japan.
H
H
Kawasaki
Y
Y
Emori
K
K
Suzuki
D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000737:chemistry*; D002154:Calpain / Q000737:chemistry*; D004789:Enzyme Activation; D004910:Erythrocyte Membrane / Q000201:enzymology; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism; D011485:Protein Binding; D011817:Rabbits
D002135:Calcium-Binding Proteins; D010446:Peptide Fragments; C033668:calpastatin; D002154:Calpain; D002118:Calcium