title: Characterization of phosphoprotein phosphatases and phosphorylase phosphatase from yeast.
abstract: Three peaks of protein phosphatase (phosphoprotein phosphohydrolase, EC 3.1.3.16) activity (fractions a, b and c) acting on muscle phosphorylase (1,4-alpha-D-glucan:orthophosphate alpha-D-glucosyltransferase, EC 2.4.1.1) were separated by DEAE-cellulose chromatography of yeast extracts. In contrast to fractions a and b, only fraction c was able to liberate phosphate from 32P-labelled inactivated yeast phosphorylase. The activity of fraction c on both substrates was totally dependent on the presence of bivalent metal ions (Mg2+, Mn2+), and was activated by Mg . ATP. Following freezing in the presence of mercaptoethanol, fractions a and b were also able to dephosphorylate yeast phosphorylase. Rabbit muscle phosphoprotein phosphatase inhibitors 1 and 2 showed that yeast phosphatases acting on muscle phosphorylase were inhibited by inhibitor 2 but not by inhibitor 1. The action of fraction c on yeast phosphorylase was not inhibited by either inhibitor. The native yeast phosphorylase phosphatase (EC 3.1.3.17) was purified 8000-fold by ion-exchange chromatography, casein-Sepharose chromatography and Sephadex G-200 gel filtration. The purified enzyme was unable to dephosphorylate rabbit muscle phosphorylase a, but acted on casein phosphate (Km 3.3 mg/ml). Molecular weight was estimated to be 78 000 and pH optimum 6.5-7.5. Activity of the enzyme was dependent on bivalent metal ions (Mg2+, Mn2+) and was inhibited by fluoride (Ki 20 mM) and succinate (Ki 10 mM).
mesh_terms: D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D008970:Molecular Weight; D010749:Phosphoprotein Phosphatases / Q000302:isolation & purification / Q000378:metabolism*; D010765:Phosphorylase Phosphatase / Q000302:isolation & purification / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*
chemical_list: D008345:Manganese; D010749:Phosphoprotein Phosphatases; D010765:Phosphorylase Phosphatase; D008274:Magnesium