title: Carboxyl-terminal truncation and site-directed mutagenesis of the EF hand structure-domain of the small subunit of rabbit calcium-dependent protease.
abstract: A mutant of the small subunit of rabbit calcium-dependent protease lacking the amino-terminal one-fourth produced in Escherichia coli could associate with the native large subunit to exert protease activity. Deletion of a few carboxyl-terminal residues of this variant small subunit caused a significant decrease in the protease activity after reconstitution with the native large subunit. Loss of the fourth EF hand loop region by further truncation of the variant small subunit made interaction with the large subunit impossible. The calcium binding assay revealed that the fourth EF hand structure of the rabbit small subunit, which has been previously demonstrated to possess two calcium-binding sites, can bind calcium ions. Furthermore it was established by site-directed mutagenesis that the first EF hand structure, in addition to the fourth one, is capable of binding calcium ions. Replacement of amino acids in the EF hand structure affected interaction with the native large subunit or the calcium sensitivity of the reconstituted product. These findings indicate that the EF hand structure-domain of the small subunit is essential for the full protease activity.
authors.affiliation: Department of Molecular Biology, Tokyo Metropolitan Institute of Medical Science.
authors.forename: Y
authors.initials: Y
authors.lastname: Minami
authors.forename: Y
authors.initials: Y
authors.lastname: Emori
authors.forename: S
authors.initials: S
authors.lastname: Imajoh-Ohmi
authors.forename: H
authors.initials: H
authors.lastname: Kawasaki
authors.forename: K
authors.initials: K
authors.lastname: Suzuki
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D002154:Calpain / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D011487:Protein Conformation; D011817:Rabbits
chemical_list: D002154:Calpain; D002118:Calcium