title: Calpastatin simultaneously binds four calpains with different kinetic constants.
abstract: Calpastatin is the endogenous, specific protein inhibitor of the calcium-dependent protease, calpain. Using an active site knock-out m-calpain mutant we have studied the enzyme's calcium-dependent binding to calpastatin by surface plasmon resonance without the complication of proteolysis. Calpastatin was capable of simultaneously binding four molecules of calpain. Its four inhibitory domains (CAST1, 2, 3, and 4) were individually expressed in Escherichia coli and the kinetics of their interaction with calpain was separately compared. Their K(d) values ranged from picomolar to nanomolar in the order CAST1>4>3>2. They have similar k(on) values but the k(off) values ranged over three orders of magnitude and can account for the differences in affinity.
authors.affiliation: Department of Biochemistry, Queen's University, Kingston, Ontario, Canada. 9rah3@qlink.queensu.ca
authors.forename: Rachel A
authors.initials: RA
authors.lastname: Hanna
authors.forename: Beatriz E
authors.initials: BE
authors.lastname: Garcia-Diaz
authors.forename: Peter L
authors.initials: PL
authors.lastname: Davies
mesh_terms: D000818:Animals; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D002154:Calpain / Q000378:metabolism*; D066298:In Vitro Techniques; D007700:Kinetics; D008954:Models, Biological; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
chemical_list: D002135:Calcium-Binding Proteins; D011994:Recombinant Proteins; C033668:calpastatin; D002154:Calpain; C449000:m-calpain