Sulfhydryl modification of the calmodulin-stimulated phosphatase, calcineurin.

TL;DRAbstract

Calcineurin (CaN), a calmodulin stimulated protein phosphatase is a heterodimer composed of a 61kD catalytic subunit and a 19kD regulatory subunit. The larger subunit has three binding sites for transition metal ions and also contains the intrinsic metal ions, Zn$\\sp{2+}$ and Fe$\\sp{3+}$. The smaller subunit contains four "EF-hand" Ca$\\sp{2+}$ binding sites. All ten of calcineurin's cysteine residues are found on the catalytic subunit. This study explores the metal ion dependence of CaN using Ca$\\sp{2+}$, Mg$\\sp{2+}$, Mn$\\sp{2+}$ and Ni$\\sp{2+}$ as well as its thiol chemistry using Ellman's reagent, iodoacetate, iodoacetamide, glutathione and a novel thiol reagent introduced by this study, 1-p-chlorophenyl-4,4-dimethyl-5-diethylamino-1-penten-3-one hydrobromide (CDDP). CaN was purified from bovine brain using DEAE-cellulose and calmodulin-agarose chromatography. Enzyme kinetics were performed with CaN in the presence of EDTA, Ca$\\sp{2+}$, Ca$\\sp{2+}$ and Mg$\\sp{2+}$, EGTA and

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