Calmodulin binding by calcineurin. Ligand-induced renaturation of protein immobilized on nitrocellulose.
The interaction of calmodulin with calcineurin, a calcium- and calmodulin-stimulated protein phosphatase, was investigated using a solid-phase assay. Binding of 125I-calmodulin by calcineurin immobilized on nitrocellulose membrane filters was of high affinity, reversible, and calcium-dependent. Complex binding kinetics reflected a time- and calcium/calmodulin-dependent conformational change of calcineurin which was shown to be ligand-induced renaturation. After renaturation and removal of calmodulin, immobilized calcineurin exhibited simple 125I-calmodulin binding kinetics with a single class of independent sites. The maximum stoichiometry of 125I-calmodulin binding to immobilized calcineurin was 0.1 mol/mol. The association rate (K1 = 8.9 x 10(3) M-1 S-1) and the dissociation rate (K-1 = 8.5 x 10(-5) s-1) yielded a dissociation constant of Kd = 10 nM. Equilibrium binding analyses gave a Kd value of 16 nM. The affinity of 125I-calmodulin for immobilized calcineurin was half that of unmodified calmodulin. Using equilibrium competition experiments, we determined, for the first time, the dissociation constant for the binding of native calmodulin by calcineurin in solution, Kd less than or equal to 0.1 nM (Kd for 125I-calmodulin = 0.23 +/- 0.09 nM). The effects of ionic strength and pH on 125I-calmodulin binding to immobilized calcineurin were characterized. The dissociation rate was dependent on free calcium concentration, with half-maximal rate at 700 nM calcium. 125I-Calmodulin equilibrium binding by the immobilized A subunit of calcineurin exhibited half the affinity of the holoenzyme, Kd = 30 nM. The described phenomenon, of reversible denaturation associated with immobilization of a protein on nitrocellulose, may be a general one open to exploitation in other systems.
Hubbard
M J
MJ
Laboratory of Biochemistry, National Cancer Institute, Bethesda, Maryland 20892.
Klee
C B
CB
D000818:Animals; D001921:Brain / Q000201:enzymology; D019703:Calcineurin; D002118:Calcium / Q000494:pharmacology; D002147:Calmodulin / Q000378:metabolism*; D002148:Calmodulin-Binding Proteins / Q000378:metabolism*; D002417:Cattle; D003101:Collodion; D004229:Dithiothreitol / Q000494:pharmacology; D004800:Enzymes, Immobilized / Q000378:metabolism*; D007700:Kinetics; D008024:Ligands; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation
D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D004800:Enzymes, Immobilized; D008024:Ligands; D003101:Collodion; D019703:Calcineurin; D010749:Phosphoprotein Phosphatases; D002118:Calcium; D004229:Dithiothreitol