Identification of high levels of type 1 and type 2A protein phosphatases in higher plants.
Extracts of Brassica napus (oilseed rape) seeds contain type 1 and type 2A protein phosphatases whose properties are indistinguishable from the corresponding enzymes in mammalian tissues. The type 1 activity dephosphorylated the beta-subunit of phosphorylase kinase selectively and was inhibited by the same concentrations of okadaic acid [IC50 (concentration causing 50% inhibition) approximately 10 nM], mammalian inhibitor 1 (IC50 = 0.6 nM) and mammalian inhibitor 2 (IC50 = 2.0 nM) as the rabbit muscle type 1 phosphatase. The plant type 2A activity dephosphorylated the alpha-subunit of phosphorylase kinase preferentially, was exquisitely sensitive to okadaic acid (IC50 approximately 0.1 nM), and was unaffected by inhibitors 1 and 2. As in mammalian tissues, a substantial proportion of plant type 1 phosphatase activity (40%) was particulate, whereas plant type 2A phosphatase was cytosolic. The specific activities of the plant type 1 and type 2A phosphatases were as high as in mammalian tissue extracts, but no type 2B or type 2C phosphatase activity was detected. The results demonstrate that the improved procedure for identifying and quantifying protein phosphatases in animal cells is applicable to higher plants, and suggests that okadaic acid may provide a new method for identifying plant enzymes that are regulated by reversible phosphorylation.
MacKintosh
C
C
Department of Biochemistry, University of Dundee, Scotland, U.K.
Cohen
P
P
D001937:Brassica / Q000201:enzymology*; D003600:Cytosol / Q000201:enzymology; D004988:Ethers, Cyclic / Q000494:pharmacology; D007476:Ionophores / Q000494:pharmacology; D019319:Okadaic Acid; D010749:Phosphoprotein Phosphatases / Q000032:analysis*; D010764:Phosphorylase Kinase / Q000378:metabolism; D010765:Phosphorylase Phosphatase / Q000037:antagonists & inhibitors
D004988:Ethers, Cyclic; D007476:Ionophores; D019319:Okadaic Acid; D010764:Phosphorylase Kinase; D010749:Phosphoprotein Phosphatases; D010765:Phosphorylase Phosphatase