Identification of protein phosphatase activities in maize seedlings.
Three phosphatases active on phosphocasein (PhosphoCasein Phosphatases) termed PCP-I, PCP-II and PCP-III were isolated from maize seedlings by DEAE-cellulose chromatography and were shown to display a different specificity toward a variety of phosphorylated substrates including pNPP, phosphohistones, phosphorylase a and several phosphopeptides containing either phosphoserine or phosphothreonine. PCP-I and PCP-II bind to heparin-Sepharose, retain a remarkable pNPP activity, are uncapable to dephosphorylate phosphorylase a, and display striking activity toward the acidic phosphopeptide AS[32P]EEEEE. They also by far prefer phosphoseryl peptide RRAS[32P]VA over its phosphothreonyl derivative and are unsensitive to okadaic acid up to 1 microM. These properties are not consistent with the belonging of PCP-I and -II to any of the known classes of protein phosphatases and suggest that they are acidic phosphatases. Conversely, PCP-III is essentially free of pNPP activity; it readily dephosphorylates phosphohistone H1 and phosphorylase a and it displays a striking preference toward the phosphothreonyl peptides (RRAT[32P]VA and RRREEET[32P]EEEAA), while the phosphoseryl peptides (RRAS[32P]VA and AS[32P]EEEEE) are very poor substrates of the enzyme. These properties together with the findings that PCP-III does not bind to heparin-Sepharose and is highly sensitive to okadaic acid (IC50 = 0.2 nM) allow to identify PCP-III with a protein phosphatase of the PP-2A class.
Jagiełło
I
I
Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland.
Donella-Deana
A
A
Szczegielniak
J
J
Pinna
L A
LA
Muszyńska
G
G
D000595:Amino Acid Sequence; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D000431:Ethanol / Q000494:pharmacology; D004988:Ethers, Cyclic / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000378:metabolism*; D008969:Molecular Sequence Data; D019319:Okadaic Acid; D010748:Phosphopeptides / Q000235:genetics / Q000378:metabolism; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013379:Substrate Specificity; D003313:Zea mays / Q000201:enzymology*
D004988:Ethers, Cyclic; D007527:Isoenzymes; D010748:Phosphopeptides; D019319:Okadaic Acid; D000431:Ethanol; D010749:Phosphoprotein Phosphatases