The Protein Phosphatases Involved in Cellular Regulation: 5. Purification and Properties of a Ca2+/Calmodulin‐Dependent Protein Phosphatase (2B) from Rabbit Skeletal Muscle

Abstract

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 G‐200 (Mr= 98000 ± 4000), chromatography on Affi‐Gel Blue and affinity chromatography on calmodulin‐Sepharose. The enzyme was purified 35000‐fold in senven days with an overall yield of 0.5%. The α‐subunit of phosphorylase kinase, protein phosphatase inhibitor‐1 and the myosin P‐light chain from rabbit skeletal muscle were dephosphorylated by protein phosphatase‐2B with similar kinetic constants. The α‐subunit of phosphorylase kinase was dephosphorylated at least 100‐fold more rapidly tha the β‐subunit, while glycogen phosphorylase, glycogen phosphorylase, glycogen synthase, histones H1 and H2B, ATP‐citrate lyase, acetyl‐CoA carboxylase, l‐pyruvate kinase and protein synthesis initiation factor eIF‐2 were not dephosporylated at significant rates. Protein phosphatase‐2B became activated 10‐fold by calmodulin (A0.5= 6nM) after chromatography on DEAE‐Sepharose and this degree of activation was maintained throughout the remainder of the purification. Calmodulin increased the Vmax of the reaction without altering the Km for inhibitor‐1. 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 Ca2+ in the presence, of 0.03 μM calmodulin. Protein phosphatase‐2B was inhibited completely by trifluoperazine; halfmaximal inhibition occurred at 45 μM in the presence of 0.03 μM calmodulin. The metabolic role of protein phosphatase‐2B in vivo is discussed in the light of the observation that this enzyme is probably identical to a major calmodulin‐binding protein of neural tissue termed calcineurin or CaAM‐BP80 [Stewart, A. A., Ingebritsen, T. S., Manalan, A., Klee, C. B., and Cohen, P. (1982) FEBS lett. 137, 80–84]. Copyright © 1983, Wiley Blackwell. All rights reserved