title: Phosphofructokinase from muscle of the marine giant barnacle Austromegabalanus psittacus: kinetic characterization and effect of in vitro phosphorylation.
abstract: The kinetic properties of phosphofructokinase from muscle of the giant cirripede Austromegabalanus psittacus were characterized, after partial purification by ion exchange chromatography on DEAE-cellulose. This enzyme showed differences regarding PFKs from other marine invertebrates: the affinity for fructose 6-phosphate (Fru 6-P) was very low, with an S(0.5) of 22.6+/-1.4 mM (mean+/-S.D., n=3), and a high cooperativity (n(H) of 2.90+/-0.21; mean+/-S.D., n=3). The barnacle PFK showed hyperbolic saturation kinetics for ATP (apparent K(m ATP)=70 microM, at 5 mM Fru 6-P, in the presence of 2 mM ammonium sulfate). ATP concentrations higher than 1 mM inhibited the enzyme. Ammonium sulfate activated the PFK several folds, increasing the affinity of the enzyme for Fru 6-P and V(max). 5'-AMP (0.2 mM) increased the affinity for Fru 6-P (S(0.5) of 6.2 mM). Fructose 2,6-bisphosphate activated the PFK, with a maximal activation at concentrations higher than 2 microM. Citrate reverted the activation of PFK produced by 0.2 mM 5'-AMP (IC(50 citrate)=2.0 mM), producing a higher inhibition than that exerted on other invertebrate PFKs. Barnacle muscular PFK was activated in vitro after exposure to exogenous cyclic-AMP (0.1 mM) as well as by phosphatidylserine (50 microg/ml), indicating a possible control by protein kinase A and a phospholipid dependent protein kinase (PKC). The results suggest a highly regulated enzyme in vivo, by allosteric mechanisms and also by protein phosphorylation.
authors.affiliation: Departamento de Acuicultura y Recursos Acuáticos, Universidad de Los Lagos, Casilla 933, Osorno, Chile. Electronic address: robert.s@ulagos.cl.
authors.forename: Robert W
authors.initials: RW
authors.lastname: Simpfendörfer
authors.affiliation: Departamento de Acuicultura y Recursos Acuáticos, Universidad de Los Lagos, Casilla 933, Osorno, Chile.
authors.forename: Karin B
authors.initials: KB
authors.lastname: Oelckers
authors.affiliation: Departamento de Acuicultura y Recursos Acuáticos, Universidad de Los Lagos, Casilla 933, Osorno, Chile.
authors.forename: Daniel A
authors.initials: DA
authors.lastname: López
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D000242:Cyclic AMP / Q000494:pharmacology; D005636:Fructosephosphates / Q000378:metabolism; D007700:Kinetics; D009132:Muscles / Q000201:enzymology*; D010718:Phosphatidylserines / Q000494:pharmacology; D025221:Phosphofructokinases / Q000302:isolation & purification / Q000378:metabolism*; D010766:Phosphorylation; D001468:Thoracica / Q000201:enzymology*
chemical_list: D005636:Fructosephosphates; D010718:Phosphatidylserines; C027618:fructose-6-phosphate; D000255:Adenosine Triphosphate; D000242:Cyclic AMP; D025221:Phosphofructokinases