Purification and characterization of a high molecular weight type 1 phosphoprotein phosphatase from the human erythrocyte.
The major Mn2+-activated phosphoprotein phosphatase of the human erythrocyte has been purified to homogeneity from the cell hemolysate. It is sensitive to both inhibitors 1 and 2 of rabbit skeletal muscle, preferentially dephosphorylates the beta subunit of the phosphorylase kinase, and dephosphorylates a broad range of substrates including phosphorylase a, p-nitro-phenyl phosphate, phosphocasein, the regulatory subunit of cyclic AMP-dependent protein kinase, and both spectrin (Km = 10 microM) and pyruvate kinase (Km = 18 microM) purified from the human erythrocyte. The purified enzyme is stimulated by Mn2+ and to a lesser extent by higher concentrations of Mg2+. The purification procedure was selected to avoid any change in molecular weight, hence subunit composition, between the crude and purified enzyme. Maintenance of the original structure is demonstrated by non-denaturing gel electrophoresis and gel filtration chromatography. Gel filtration of the purified holoenzyme shows a single active component with a Stokes radius of 58 A at a molecular weight position of 180,000. Sedimentation velocity in a glycerol gradient gives a value of 6.1 for S20, w. Together these data indicate a molecular weight of about 135,000. Two bands of equal intensity appear on sodium dodecyl sulfate-gel electrophoresis at molecular weights of 61,700 and 36,300, suggesting a subunit composition of two 36,000 and one 62,000 subunits. The 36-kDa catalytic subunit can be isolated by freezing and thawing the holoenzyme or by hydrophobic chromatography of the holoenzyme. The catalytic subunit shows unchanged substrate and inhibitor specificity but altered metal ion activation.
D000818:Animals; D002352:Carrier Proteins; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004722:Endoribonucleases; D004789:Enzyme Activation; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D007527:Isoenzymes / Q000097:blood / Q000302:isolation & purification*; D007700:Kinetics; D046911:Macromolecular Substances; D008345:Manganese / Q000378:metabolism; D008970:Molecular Weight; D009132:Muscles / Q000032:analysis; D010749:Phosphoprotein Phosphatases / Q000097:blood / Q000302:isolation & purification*; D010764:Phosphorylase Kinase / Q000378:metabolism; D011506:Proteins / Q000494:pharmacology; D016601:RNA-Binding Proteins; D011817:Rabbits; D000097847:Protein Phosphatase Inhibitory Proteins
D002352:Carrier Proteins; D004722:Endoribonucleases; D047908:Intracellular Signaling Peptides and Proteins; D007527:Isoenzymes; D046911:Macromolecular Substances; D008345:Manganese; D010749:Phosphoprotein Phosphatases; D010764:Phosphorylase Kinase; D011506:Proteins; D016601:RNA-Binding Proteins; C021042:protein phosphatase inhibitor-1; C024833:protein phosphatase inhibitor-2; D000097847:Protein Phosphatase Inhibitory Proteins; C097081:PPP1R8 protein, human