title: Partial purification and characterization of two phosvitin phosphatases from rat brain.
abstract: The mechanism of dephosphorylation of multiphosphorylated proteins in the brain is not well understood. We have used the multiphosphorylated protein, phosvitin as a model substrate and undertaken the purification and characterization of brain phosphatases that preferentially dephosphorylate multiphosphorylated proteins. Two phosvitin phosphatase activities, termed Phosvitin Phosphatase 1 and 2 (PvP1, PvP2), which show acidic pH optima were resolved from the 33,000g supernatant fraction from rat brain by a procedure employing successive DEAE-cellulose, Sepharose 6B, second DEAE-cellulose and FPLC/Superose 6 chromatography steps. Following FPLC/Superose 6 size exclusion chromatography of PvP1 and PvP2, single peaks of phosvitin phosphatase activities were eluted in the range of 160-220 kDa with acidic pH optima. When FPLC/Sepharose 6 chromatography was performed in the presence of 0.5 M NaCl and 0.1% Triton X-100, low molecular mass protein phosphatase forms were produced in addition to the high-M, activity peak, ranging from 25 to 35 kDa (PvP1) and from 15 to 25 kDa (PvP2). Under these conditions, both high- and low-M, forms of PvP1 and PvP2 exhibited neutral pH optima. Both phosphatases dephosphorylate also (i) phosphorylase a, (ii) the alpha and beta subunits of phosphorylase kinase, and (iii) the microtubule-associated protein tau, phosphorylated by cAMP-dependent protein kinase. The present results suggest that two forms of protein phosphatases, displayed molecular and biochemical characteristics both similar and distinct from type 1 and type 2A protein phosphatases, are present in rat brain.
authors.affiliation: Institute of Biological Research and Biotechnology, National Hellenic Research Foundation, Athens, Greece.
authors.forename: T P
authors.initials: TP
authors.lastname: Geladopoulos
authors.forename: T G
authors.initials: TG
authors.lastname: Sotiroudis
authors.forename: A E
authors.initials: AE
authors.lastname: Evangelopoulos
mesh_terms: D000818:Animals; D001921:Brain / Q000201:enzymology*; D003124:Colorimetry; D004396:Coloring Agents; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D009419:Nerve Tissue Proteins / Q000302:isolation & purification* / Q000378:metabolism*; D010749:Phosphoprotein Phosphatases / Q000302:isolation & purification*; D010766:Phosphorylation; D054645:Protein Phosphatase 1; D054648:Protein Phosphatase 2; D051381:Rats; D017208:Rats, Wistar; D012394:Rosaniline Dyes; D012680:Sensitivity and Specificity; D016875:tau Proteins / Q000378:metabolism
chemical_list: D004396:Coloring Agents; D009419:Nerve Tissue Proteins; D012394:Rosaniline Dyes; D016875:tau Proteins; C005095:malachite green; C099391:phosvitin phosphatase 1; C099392:phosvitin phosphatase 2; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; D054648:Protein Phosphatase 2