Calcineurin, a heterodimer of calcineurin B, a 19,000 Mr Ca2+-binding subunit, and calcineurin A, a 61,000 Mr calmodulin-binding subunit, was previously proposed to be a calmodulin-and Ca2+-regulated protein phosphatase. Like other calmodulin-stimulated enzymes, calcineurin can be activated and rendered calmodulin-and Ca2+-independent by limited proteolysis. By glycerol gradient centrifugation, the native enzyme has a s2o w of 4.5 S in EGTA and 5 S in the presence of Ca2~+calmodulin. capable of binding four Ca2" (2). Recently, a Ca2+-and calmodulin-regulated phosphoprotein phosphatase activity has been found to be associated with calcineurin (5, 6). Other calmodulin-stimulated protein phosphatases, the "protein phosphatases 2B," have been detected in a large number of tissues (7). The skeletal muscle enzyme, which has been purified to homogeneity, has a subunit composition similar to that of calcineurin (8). A calcineurin-like protein is also present at low levels in heart (9, 10). Whereas calcineurin and the calmodulin-regulated protein phosphatases from different tissues share similar enzymatic activities, they exhibit differences in the molecular weight of their large subunits. They may also differ antigenically, because calcineurin antibody appears to be relatively specific for the brain protein (11 MATERIALS AND METHODS Calcineurin, purified from bovine brain as described (6), was dialyzed overnight at 40C against 0.04 M Tris HCI, pH 7.5, containing 0.1 M NaCl, 1 mM MgCl2, 0.1 mM EGTA, and 0.2 mM dithiothreitol in the presence or absence of 5% (vol/vol) glvcerol prior to tryptic digestion or enzymatic assay. Calmodulin was purified from bovine testes by the method of Autric et al.(12) with modifications (unpublished results). '"I-Labeled calmodulin ('"I-calmodulin) (specific activity 100 Ci/mmol; 1 Ci = 3.7 X 10'0 Bq) was prepared as described (6). Dephosphorylated smooth muscle myosin light chains, prepared from chicken gizzard by the method of Perrie and Perry (13) ,ug/ml. Reactions were stopped by addition of soybean trypsin inhibitor at 100 gg/ml and reaction mixtures were cooled on ice.Phosphatase activity was assayed in 50 ,l of 0.02 M Tris HCl buffer, pH 8.0, containing 0.1 M NaCl, 6 mM MgCl2, bovine serum albumin at 0.1 mg/ml, 10 puM MnCl2, 0.5 mnM dithiothreitol, 0.3-0.8 ,ug of calcineurin, and 1 ,.M [32P]phosphorylated myosin light chains (106 cpm/nmol). Calmodulin (1 p.M), 4291 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
