The Protein Phosphatases Involved in Cellular Regulation.

Protein phosphatase-2B was purified from extracts of rabbit skeletal muscle by a procedure that involved fractionation with ammonium sulphate, chromatography on DEAE-Sepharose, fractionation with poly(ethylene glycol), gel filtration on Sephadex 0-200 (M_r = 98000 ± 4000). chromatography on Affi-Gel Blue and affinity chromatography on calmodulin-Sepharose. The enzyme was purified 3500-fold in seven days with an overall yield of 0.5%. The α-subunit of phosphorylase kinase, protein phosphatase inhibitor-i and the myosin P-Light chain from rabbit skeletal muscle were dephosphorylated by protein phosphatase-2B with similar kinetic constants. The &alpha-subunit of phosphorylase kinase was dephosphorylated at least 100-fold more rapidly than the β-subunit, while glycogen phosphorylase, glycogen synthase, histones H1 and H2B. ATP-citrate lyase, acetyl-CoA carboxylase, L-pyruvate kinase and protein synthesis initiation factor elF-2 were not dephosphorylated at significant rates. Protein phosphatase-2B became activated 10-fold by calmodulin (A_0.5 = 6 nM) after chromatography on DEAE-Sepharose and this degree of activation was maintained throughout the remainder of the purification. Calmodulin increased the V_max of the reaction without altering the K_m for inhibitor-I. The activity of protein phosphatase-2B was completely dependent on Ca²⁺ in the presence or absence of calmodulin. Half-maximal activation was observed at 1.0 μM Ca^{2 +} in the absence, and at 0.5 μM Ca²⁺ in the presence, of 0.03 μM calmodulin. Protein phosphatase-2B was inhibited completely by trifluoperazine; half- maximal inhibition occurred at 45 μM in the absence and 35 μM in the presence of 0.03 μM calmodulin. The metabolic rote of protein phosphatase-2B in vivo is discussed in the light of the observation that this enzyme is probably identical to a major calmodulinabinding protein of neural tissue termed calcineurin or CaM-BP₈₀ (Stewart, A. A., Ingebritsen, T. S., Manalan, A., Klee, C. B., and Cohen, P. (1982) FEBS Let!. 137, 80-84]. [ABSTRACT FROM AUTHOR]