Partial purification and characterization of two phosvitin phosphatases from rat brain.
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.
Geladopoulos
T P
TP
Institute of Biological Research and Biotechnology, National Hellenic Research Foundation, Athens, Greece.
Sotiroudis
T G
TG
Evangelopoulos
A E
AE
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
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