ESR STUDY ON CALCINEURIN

X-band electron spin resonance spectroscopy was used to investigate th e binding of Mn2+ to the apo-forms of calcineurin and its A and B subu nits. The results indicated the presence of 2 Mn2+ binding sites of di fferent affinities (20 mu mol/L and 60 mu mol/L) in the calcineurin A subunit and Mn2+ binding sites in the calcineurin subunit B, 2 high af finity and 2 low affinity binding sites with K-d's of 4 mu mol/L and 9 0 mu mol/L, respectively. Interestingly and suite surprisingly, Mn2+ b inding to the holoenzyme was characterized by only 2 binding sites wit h K-d's of 7 mu mol/L and 33 mu mol/L. However, in the presence of cal modulin about 10 Mn2+ sites were detected, and the Mn2+ calmodulin-cal cineurin complex exhibited enzymatic activity. These results, based on direct spectral measurements of the metal ligand, demonstrate that Mn 2+ binds to both free subunits of calcineurin in a manner distinct fro m binding to the holoenzyme. Also, the data suggest that conformationa l changes occur upon heterodimer formation and association of the holo enzyme with the regulatory protein calmodulin.