title: Purification and partial characterization of protein phosphatases from rat thymus.
abstract: Protein phosphatases assayed with phosphorylase alpha are present in the soluble and particulate fractions of rat thymocytes. Phosphorylase phosphatase activity in the cytosol fraction was resolved by heparin-Sepharose chromatography into type-1 and type-2A enzymes. Similarities between thymocyte and muscle or liver protein phosphatase-1 included preferential dephosphorylation of the beta subunit of phosphorylase kinase, inhibition by inhibitor-2 and retention by heparin-Sepharose. Similarities between thymocyte and muscle or liver protein phosphatase-2A included specificity for the alpha subunit of phosphorylase kinase, insensitivity to the action of inhibitor-2, lack of retention by heparin-Sepharose and stimulation by polycationic macromolecules such as polybrene, protamine and histone H1. Protein phosphatase-1 from the cytosol fraction of thymocytes had an apparent molecular mass of 120 kDa as determined by gel filtration. The phosphatase-2A separated from the cytosol of thymocytes may correspond to phosphatase-2A0, since it was completely inactive (latent) in the absence of polycation and had activity only in the presence of polycations. The apparent molecular mass of phosphatase-2A0 from thymocytes was 240 kDa as determined by gel filtration. The catalytic subunit of thymocyte type-1 protein phosphatase was purified with heparin-Sepharose chromatography followed by gel filtration and fast protein liquid chromatography on Mono Q column. The purified type-1 catalytic subunit exhibited a specific activity of 8.2 U/mg and consisted of a single protein of 35 kDa as judged by SDS-gel electrophoresis. The catalytic subunit of type-2A phosphatase from thymocytes appearing in the heparin-Sepharose flow-through fraction was further purified on protamine-Sepharose, followed by gel filtration. The specific activity of the type-2A catalytic subunit was 2.1 U/mg and consisted of a major protein of 34.5 kDa, as revealed by SDS-gel electrophoresis.
authors.affiliation: Department of Medical Chemistry, University School of Medicine, Debrecen Hungary.
authors.forename: E
authors.initials: E
authors.lastname: Bakó
authors.forename: V
authors.initials: V
authors.lastname: Dombrádi
authors.forename: F
authors.initials: F
authors.lastname: Erdödi
authors.forename: L
authors.initials: L
authors.lastname: Zumo
authors.forename: P
authors.initials: P
authors.lastname: Kertai
authors.forename: P
authors.initials: P
authors.lastname: Gergely
mesh_terms: D000818:Animals; D002845:Chromatography; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003600:Cytosol / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D008099:Liver / Q000201:enzymology; D008970:Molecular Weight; D009132:Muscles / Q000201:enzymology; D010749:Phosphoprotein Phosphatases / Q000302:isolation & purification*; D010764:Phosphorylase Kinase / Q000378:metabolism; D011073:Polyamines; D000071228:Polyelectrolytes; D011108:Polymers / Q000494:pharmacology; D054645:Protein Phosphatase 1; D054648:Protein Phosphatase 2; D011817:Rabbits; D051381:Rats; D013379:Substrate Specificity; D013950:Thymus Gland / Q000201:enzymology* / Q000648:ultrastructure
chemical_list: D010749:Phosphoprotein Phosphatases; D010764:Phosphorylase Kinase; D011073:Polyamines; D000071228:Polyelectrolytes; D011108:Polymers; D054645:Protein Phosphatase 1; D054648:Protein Phosphatase 2