Identification of two isoenzymes of protein phosphatase 2C in both rabbit skeletal muscle and liver

Abstract

Protein phosphatase 2C was isolated from rabbit skeletal muscle by a procedure that involved chromatography on DEAE‐cellulose, precipitation with ammonium sulphate, gel‐filtration on Sephadex G‐100, affinity chromatography on thiophosphorylated myosin‐P‐light‐chain–Sepharose and chromatography on Mono Q. The enzyme was purified about 35000‐fold and 0.3–0.4 mg was isolated from 2500 g skeletal muscle within 5 days. The final step resolved the activity into two peaks, termed protein phosphatases 2C₁ and 2C₂, that possessed identical substrate specificities and enzymatic properties. About 2.5‐fold more protein phosphatase 2C₂ was isolated than protein phosphatase 2C₁. Protein phosphatases 2C₁ and 2C₂ migrated as single bands on SDS/polyacrylamide gels yielding apparent molecular masses of 44 kDa and 42 kDa, respectively, and the native proteins were both monomeric at pH 7.5 as judged by their elution from Sephadex G‐100 and Sephacryl S200. Peptide maps of protein phosphatases 2C₁ and 2C₂, obtained after separate digestions with four different proteinases, were different, indicating that they are isoenzymes. Protein phosphatases 2C₁ and 2C₂ were purified from rabbit liver by the same procedure, and 0.2 mg (2C₁+ 2C₂) was isolated from 120 g hepatic tissue. Hepatic protein phosphatases 2C₁ and 2C₂ were also isolated in a molar ratio of about 1:2.5, and their enzymatic properties and apparent molecular masses in the presence and absence of SDS were identical to the skeletal muscle enzymes. Protein phosphatases 2C₄ from muscle and liver displayed identical peptide maps, as did protein phosphatases 2C₂ from these two tissues. It is concluded that the same two isoenzymes of protein phosphatase 2C are present in skeletal muscle and liver.