title: Profiling of calpain activity with a series of FRET-based substrates.
abstract: Calpains are intracellular proteases that selectively cleave proteins in response to calcium signals. Although calpains cut many different sequences, residue preferences within peptide substrates were recently determined and incorporated into a superior FRET (fluorescence resonance energy transfer)-based substrate (PLFAER). Here we show PLFAER is cleaved by calpain at the intended F-A scissile bond. Sequential replacement of individual residues by alanine reduced activity except with PLFAAR, which is cleaved 2.3 times faster than PLFAER. The rates of hydrolysis of the alanine-substituted substrates were used to compare substrate preferences of calpain, papain and cathepsins B and L. The preferences of the two major isoforms, calpains 1 and 2, were virtually indistinguishable and were very similar to those of the calpain 1 protease core and papain. However, the activity profiles with the FRET substrate series were significantly different for the cathepsins, particularly cathepsin B.
authors: Department of Biochemistry, Queen's University, Kingston, ON, Canada K7L 3N6.
authors: Jacqueline C
authors: JC
authors: Kelly
authors: Dominic
authors: D
authors: Cuerrier
authors: Laurie A
authors: LA
authors: Graham
authors: Robert L
authors: RL
authors: Campbell
authors: Peter L
authors: PL
authors: Davies
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D002154:Calpain / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain / Q000235:genetics; D002403:Cathepsins / Q000737:chemistry / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes; D006868:Hydrolysis; D066298:In Vitro Techniques; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D009282:Naphthalenesulfonates; D009842:Oligopeptides / Q000737:chemistry; D010206:Papain / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D004124:p-Dimethylaminoazobenzene / Q000031:analogs & derivatives
chemical_list: D005456:Fluorescent Dyes; D007527:Isoenzymes; D009282:Naphthalenesulfonates; D009842:Oligopeptides; D011994:Recombinant Proteins; C063126:5-((2-aminoethyl)amino)naphthalene-1-sulfonic acid; C063127:4-(4-dimethylaminophenylazo)benzoic acid; D004124:p-Dimethylaminoazobenzene; D002403:Cathepsins; D002154:Calpain; D010206:Papain