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 (Mr) of 225,000 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 polylysine-agarose, 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, alpha and beta, with Mr of 40,000 and 75,000, respectively. These values, in conjunction with the native Mr and the molar ratios of the subunits estimated by densitometric analysis of the gel, suggested that the subunit composition of the enzyme is alpha 2 beta 2. When treated with 1.7% (v/v) 2-mercaptoethanol at -20 degrees C or with ethanol, the enzyme released the catalytic alpha subunit of Mr 40,000. The protein phosphatase was activated by basic proteins e.g. protamine (A 0.5 = 1 microM), histone H1 (A 0.5 = 1.6 microM) and polylysine (A 0.5 = 0.2 microM) and inhibited by ATP (I 0.5 = 12 microM), 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 substrates used by the enzyme. The function of reversible phosphorylation-dephosphorylation of mRNP as a regulatory mechanism in mRNP metabolism is discussed.
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D001156:Artemia / Q000201:enzymology*; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D006657:Histones / Q000378:metabolism; D046911:Macromolecular Substances; D008970:Molecular Weight; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D011107:Polylysine / Q000378:metabolism; D011479:Protamines / Q000378:metabolism; D012261:Ribonucleoproteins / Q000378:metabolism*; D012969:Sodium Fluoride / Q000494:pharmacology; D013696:Temperature
D006657:Histones; D046911:Macromolecular Substances; D011479:Protamines; D012261:Ribonucleoproteins; C026452:messenger ribonucleoprotein; D011107:Polylysine; D000255:Adenosine Triphosphate; D012969:Sodium Fluoride; D010749:Phosphoprotein Phosphatases