Partial purification and characterization of a type 1 protein phosphatase in purified nuclei of pea plumules.
We report the isolation and characterization of a protein Ser/Thr phosphatase from highly purified pea nuclei. In subnuclear fractions, more than 75% of Ser/Thr protein phosphatase activity was associated with the chromatin fraction, whereas the other 25% was in the nuclear membrane/nucleoplasmic fraction when phosphorylase a was used as a substrate. The enzyme was purified approx. 2750-fold to a specific activity of approx. 4000 nmol/min per mg. The molecular mass of the enzyme was 34 kDa as estimated by molecular sieve chromatography, and approx. 40 kDa as estimated by SDS/PAGE. The phosphatase was inhibited by okadaic acid with an IC50 of approx. 15 nM, by rabbit muscle inhibitor 2 with an IC50 of approx. 10 nM, and by microcystin-LR with an IC50 of approx. 0.05 nM. The enzyme did not require Ca2+, Mg2+ or Mn2+ for its activity; instead, these cations showed some inhibitory effects. It was inhibited by NaF or citrate but not by tartrate, molybdate or vanadate under the conditions tested. Its sensitivities towards the various phosphatase inhibitors and its substrate specificity were very similar to those characteristic of the type I Ser/Thr protein phosphatases well studied in animal systems. The enzyme was able to selectively dephosphorylate a 92 kDa nuclear protein that had been phosphorylated by one or more endogenous protein kinases.
Guo
Y L
YL
Department of Botany, University of Texas at Austin 78713, USA.
Roux
S J
SJ
D000818:Animals; D002467:Cell Nucleus / Q000201:enzymology*; D002843:Chromatin / Q000201:enzymology; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D007525:Isoelectric Focusing; D008970:Molecular Weight; D009687:Nuclear Proteins / Q000378:metabolism; D018532:Pisum sativum / Q000201:enzymology*; D010749:Phosphoprotein Phosphatases / Q000302:isolation & purification* / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism; D011817:Rabbits; D013379:Substrate Specificity
D002843:Chromatin; D009687:Nuclear Proteins; D010750:Phosphoproteins; D010749:Phosphoprotein Phosphatases