title: Characterization of the major phosphofructokinase-dephosphorylating protein phosphatases from Ascaris suum muscle.
abstract: In contrast to the mammalian enzyme, PFK from the nematode Ascaris suum is activated following phosphorylation (Daum et al. (1986) Biochem. Biophys. Res. Commun. 139, 215-221) catalyzed by a cAMP-dependent protein kinase (Thalhofer et al. (1988) J. Biol. Chem. 263, 952-957). In the present report, we describe the characterization of the major PFK dephosphorylating phosphatases from Ascaris muscle. Two of these phosphatases exhibit apparent M(r) values of 174,000 and 126,000, respectively, and are dissociated to active 33 kDa proteins by ethanol precipitation. Denaturing electrophoresis of each of the enzyme preparations showed two bands of M(r) 33,000 and 63,000. The enzymes are classified as type 2A phosphatases according to their inhibition by subnanomolar concentrations of okadaic acid, the lack of inhibition by heat-stable phosphatase inhibitors 1 and 2, and their preference for the alpha- rather than for the beta-subunit of phosphorylase kinase. Like other type 2A phosphatases, they exhibit broad substrate specificities, are activated by divalent cations and polycations, and inhibited by fluoride, inorganic phosphate and adenine nucleotides. In addition, we have found that PFK is also dephosphorylated by an unusual protein phosphatase. This exhibits kinetic properties similar to type 2A protein phosphatases, but has a distinctly lower sensitivity towards inhibition by okadaic acid (IC50 approx. 20 nM). Partial purification of the enzyme provided evidence that it is composed of a 30 kDa catalytic subunit and probably two other subunits (molecular masses 66 and 72 kDa). The dephosphorylation of PFK by protein phosphatases is strongly inhibited by heparin. This effect, however, is substrate-specific and does not occur with Ascaris phosphorylase a.
authors.affiliation: Faculty of Biology, University of Konstanz, Germany.
authors.forename: G
authors.initials: G
authors.lastname: Daum
authors.forename: B
authors.initials: B
authors.lastname: Schmid
authors.forename: C
authors.initials: C
authors.lastname: MacKintosh
authors.forename: P
authors.initials: P
authors.lastname: Cohen
authors.forename: H W
authors.initials: HW
authors.lastname: Hofer
mesh_terms: D000818:Animals; D001200:Ascaris / Q000201:enzymology*; D006493:Heparin / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000032:analysis / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D010766:Phosphorylation
chemical_list: D006493:Heparin; C031915:phosphofructokinase phosphatase; D010749:Phosphoprotein Phosphatases