title: m-Calpain subunits remain associated in the presence of calcium.
abstract: The hypothesis that calpain subunits dissociate in the presence of Ca2+ has been tested by methods which avoid interference by Ca2+-induced aggregation and large subunit autolysis. Inactive Cys105Ser-m-calpain, bound either to Ni-NTA-agarose or to immobilized casein, after incubation with Ca2+, could be recovered in high yield as a heterodimer. Natural bovine m-calpain, after irreversible inhibition with Z-LLY-CHN2, also bound to immobilized casein and was eluted as a heterodimer. The Ca2+ requirements of calpain containing a small subunit with EF-hand mutations were higher, both before and after autolysis, than those of wild-type calpain. In mixtures of wild-type and mutant enzymes, subunit exchange did not occur in the presence of Ca2+. The results demonstrate that the subunits in both natural and recombinant m-calpain, in the given experimental conditions, remain associated in the presence of Ca2+ both before and after autolysis.
authors.affiliation: Department of Biochemistry, Queen's University, and The Protein Engineering Network of Centres of Excellence, Kingston, Ont., Canada.
authors.forename: P
authors.initials: P
authors.lastname: Dutt
authors.forename: J S
authors.initials: JS
authors.lastname: Arthur
authors.forename: D E
authors.initials: DE
authors.lastname: Croall
authors.forename: J S
authors.initials: JS
authors.lastname: Elce
mesh_terms: D000409:Alanine; D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D002154:Calpain / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism; D002364:Caseins / Q000378:metabolism; D020134:Catalytic Domain; D002417:Cattle; D003545:Cysteine; D019281:Dimerization; D018698:Glutamic Acid; D007700:Kinetics; D046911:Macromolecular Substances; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D012694:Serine
chemical_list: D002364:Caseins; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D018698:Glutamic Acid; D012694:Serine; D002154:Calpain; D003545:Cysteine; D000409:Alanine; D002118:Calcium