Phosphofructokinase from muscle of the marine giant barnacle Austromegabalanus psittacus: kinetic characterization and effect of in vitro phosphorylation.
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.
Simpfendörfer
Robert W
RW
Departamento de Acuicultura y Recursos Acuáticos, Universidad de Los Lagos, Casilla 933, Osorno, Chile. Electronic address: robert.s@ulagos.cl.
Oelckers
Karin B
KB
Departamento de Acuicultura y Recursos Acuáticos, Universidad de Los Lagos, Casilla 933, Osorno, Chile.
López
Daniel A
DA
Departamento de Acuicultura y Recursos Acuáticos, Universidad de Los Lagos, Casilla 933, Osorno, Chile.
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*
D005636:Fructosephosphates; D010718:Phosphatidylserines; C027618:fructose-6-phosphate; D000255:Adenosine Triphosphate; D000242:Cyclic AMP; D025221:Phosphofructokinases