Activation of calcineurin by the trivalent metal terbium.
As a possible probe for metal activation of calcineurin, Tb3+ was tested for effects on calcineurin activity. Calcineurin was activated by Tb3+ with the following kinetic parameters estimated: k(cat) = 0.78 +/- 0.02 sec(-1), Km(pNPP) = 32.6 +/- 1.8 mM, and K(act)(Tb3+) = 0.08 +/- 0.03 mM. Terbium luminescence was demonstrated in the presence of the heterodimer of calcineurin and exploited to localize the binding of exogenous metal to the enzyme active site. Exogenous Mn2+ reduced luminescence, although the affinity of calcineurin for Tb3+ seemed to be greater. Putative active-site ligands, such as para-nitrophenol and a synthetic peptide from the autoinhibitory region, reduced the luminescence of terbium. Collectively, these data suggested that Tb3+ was binding directly at the active site of calcineurin, with the corollary that exogenous activating metal (Mn2+) binds at the active site of the enzyme. These data support the hypothesis that activating, exogenous divalent metal participates directly in catalysis.
Martin
B L
BL
Department of Biochemistry, University of Tennessee, Memphis 38163, USA. bmartin@utmem1.utmem.edu
Jurado
L A
LA
D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000201:enzymology; D019703:Calcineurin / Q000378:metabolism*; D002417:Cattle; D019281:Dimerization; D004789:Enzyme Activation; D007700:Kinetics; D008163:Luminescent Measurements; D011485:Protein Binding; D013725:Terbium / Q000378:metabolism / Q000494:pharmacology*
D013725:Terbium; D019703:Calcineurin