title: Studies of the active site of m-calpain and the interaction with calpastatin.
abstract: Calpain autolyses in the presence of Ca2+. In the case of m-calpain (80 + 30 kDa) the first product is an 80 + 18 kDa species which has an intact large subunit and the C-terminal Ca(2+)-binding domain of the small subunit. It was possible to bind E64 into the active site of calpain in the presence of Ca2+ before cleavage of either calpain subunit. This suggests that the active site is functional before any autolysis has occurred and that calpain is not a proenzyme. Prolonged autolysis generates several fragments including a 42 kDa active-site domain fragment that showed no proteolytic activity and Ca(2+)-binding domain fragments. Some of the Ca(2+)-binding domain fragments were found to exist as heterodimers (23 + 18 kDa and 22 + 18 kDa), with the Ca(2+)-binding domain of the large subunit interacting with the Ca(2+)-binding domain of the small subunit. These species were true heterodimers, as they showed co-elution of the two Ca(2+)-binding domains on ion-exchange and gel-filtration chromatography, and immunoprecipitation of both polypeptides with an antiserum specific for the small-subunit Ca(2+)-binding domain. The generation of the dimer species after only 15 min autolysis suggests that the interaction between the Ca(2+)-binding domains is present in the native calpain structure. The interaction of calpain with calpastatin was investigated using an assay based on binding to calpastatin-Sepharose and a competitive binding assay. Calpain, active-site-blocked calpain and calpain fragments generated by autolysis were studied. Calpain bound to calpastatin in the presence of Ca2+; however, the isolated active-site-containing 80 kDa large subunit (proteolytically inactive), a 42 kDa active-site-containing fragment (proteolytically inactive) and Ca(2+)-binding domain fragments of calpain did not. Active-site-blocked calpain bound to calpastatin, but with an affinity reduced by approximately two orders of magnitude when compared with native calpain.
authors.affiliation: Laboratory of Molecular Biophysics, University of Oxford, U.K.
authors.forename: C
authors.initials: C
authors.lastname: Crawford
authors.forename: N R
authors.initials: NR
authors.lastname: Brown
authors.forename: A C
authors.initials: AC
authors.lastname: Willis
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D002154:Calpain / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D002846:Chromatography, Affinity; D015853:Cysteine Proteinase Inhibitors / Q000302:isolation & purification / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D007668:Kidney / Q000201:enzymology*; D046911:Macromolecular Substances; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009206:Myocardium / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D013552:Swine
chemical_list: D002135:Calcium-Binding Proteins; D015853:Cysteine Proteinase Inhibitors; D046911:Macromolecular Substances; D010446:Peptide Fragments; C033668:calpastatin; D002154:Calpain