title: m-Calpain activation in vitro does not require autolysis or subunit dissociation.
abstract: Calpains are Ca(2+)-dependent, intracellular cysteine proteases involved in many physiological functions. How calpains are activated in the cell is unknown because the average intracellular concentration of Ca(2+) is orders of magnitude lower than that needed for half-maximal activation of the enzyme in vitro. Two of the proposed mechanisms by which calpains can overcome this Ca(2+) concentration differential are autoproteolysis (autolysis) and subunit dissociation, both of which could release constraints on the core by breaking the link between the anchor helix and the small subunit to allow the active site to form. By measuring the rate of autolysis at different sites in calpain, we show that while the anchor helix is one of the first targets to be cut, this occurs in the same time-frame as several potentially inactivating cleavages in Domain III. Thus autolytic activation would overlap with inactivation. We also show that the small subunit does not dissociate from the large subunit, but is proteolyzed to a 40-45k heterodimer of Domains IV and VI. It is likely that this autolysis-generated heterodimer has previously been misidentified as the small subunit homodimer produced by subunit dissociation. We propose a model for m-calpain activation that does not involve either autolysis or subunit dissociation.
authors.affiliation: Department of Biochemistry, Queen's University, Kingston, ON, Canada K7L 3N6.
authors.forename: Jordan S
authors.initials: JS
authors.lastname: Chou
authors.forename: Francis
authors.initials: F
authors.lastname: Impens
authors.forename: Kris
authors.initials: K
authors.lastname: Gevaert
authors.forename: Peter L
authors.initials: PL
authors.lastname: Davies
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D002154:Calpain / Q000737:chemistry* / Q000378:metabolism; D020134:Catalytic Domain; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D008954:Models, Biological; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D055503:Protein Multimerization / Q000187:drug effects; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D051381:Rats; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D021122:Protein Subunits; D002154:Calpain; C449000:m-calpain; D002118:Calcium