Purification, subunit structure and properties of a high‐molecular‐mass protein phosphatase capable of dephosphorylating mRNP of the brine shrimp Artemia sp.

A phosphoprotein phosphatase active towards casein, phosphorylase a and mRNP proteins has been detected in the cytosol of cryptobiotic gastrulae of Artemia sp. This phosphatase has a relative molecular mass ( M r ) of 225000 as measured by gel filtration on Sephadex G‐200 and has been purified to near homogeneity by ion‐exchange chromatography on different DEAE‐substituted matrices, affinity chromatography on polylysineagarose, histone‐Sepharose 4B and protamine‐agarose, hydrophobic chromatography on phenyl‐Sepharose 4B and gel filtration on Sephadex G‐200. Sodium dodecyl sulphate gel electrophoresis of the final purification step revealed that the enzyme contains two types of subunits, α and β, with M r of 40 000 and 75 000, respectively. These values, in conjunction with the native M r and the molar ratios of the subunits estimated by densitometric analysis of the gel, suggested that the subunit composition of the enzyme is α 2 β 2 . When treated with 1.7% (v/v) 2‐mercaptoethanol at ‐20°C or with ethanol, the enzyme released the catalytic α subunit of M r 40 000. The protein phosphatase was activated by basic proteins e.g. protamine ( A 0.5 = 1 μM), histone H1 ( A 0.5 = 1.6 μM) and polylysine ( A 0.5 = 0.2 μM) and inhibited by ATP ( I 0.5 = 12 μM), NaF ( I 0.5 = 3.1 mM) and pyrophosphate ( I 0.5 = 0.6 mM). The enzyme is a polycation‐stimulated protein phosphatase. Purified mRNP proteins, phosphorylated by the mRNP‐associated casein kinase type II, are among the substratcs used by the enzyme. The function of reversible phosphorylation‐dephosphorylation of mRNP as a regulatory mechanism in mRNP metabolism is discussed.