ESR Study on calcineurin

【摘要】 <正> X-band electron spin resonance spectroscopy was used to investigate the binding of Mn²⁺tothe apo-forms of calcineurin and its A and B subunits.The results indicated the presence of 2Mn²⁺binding sites of different affinities（20μmol/L and 60μmol/L）in the calcineurin A subunit and 4Mn²⁺binding sites in the calcineurin subunit B,2 high affinity and 2 low affinity binding sites withK_d’s of 4μmol/L and 90μmol/L,respectively.Interestingly and quite surprisingly,Mn²⁺binding to theholoenzyme was characterized by only 2 binding sites with K_d’s of 7μmol/L and 33μmol/L.However,inthe presence of calmodulin about 10 Mn²⁺sites were detected,and the Mn²⁺calmodulin-calcineurin complexexhibited enzymatic activity.These results,based on direct spectral measurements of the metal ligand,demonstrate that Mn²⁺binds to both free subunits of calcineurin in a manner distinct from binding to theholoenzyme.Also,the data suggest that conformational changes occur upon heterodimer formation andassociation of the holoenzyme with the regulatory protein calmodulin.更多还原

【Abstract】 X-band electron spin resonance spectroscopy was used to investigate the binding of Mn²⁺to the apo-forms of calcineurin and its A and B subunits.The results indicated the presence of 2 Mn²⁺binding sites of different affinities（20μmol/L and 60μmol/L）in the calcineurin A subunit and 4 Mn²⁺binding sites in the calcineurin subunit B,2 high affinity and 2 low affinity binding sites with K_d’s of 4μmol/L and 90μmol/L,respectively.Interestingly and quite surprisingly,Mn²⁺binding to the holoenzyme was characterized by only 2 binding sites with K_d’s of 7μmol/L and 33μmol/L.However,in the presence of calmodulin about 10 Mn²⁺sites were detected,and the Mn²⁺calmodulin-calcineurin complex exhibited enzymatic activity.These results,based on direct spectral measurements of the metal ligand, demonstrate that Mn²⁺binds to both free subunits of calcineurin in a manner distinct from binding to the holoenzyme.Also,the data suggest that conformational changes occur upon heterodimer formation and association of the holoenzyme with the regulatory protein calmodulin.更多还原