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Journal of Biochemistry Advance Access originally published online on May 15, 2008
Journal of Biochemistry 2008 144(2):235-244; doi:10.1093/jb/mvn066
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© 2008 The Japanese Biochemical Society

Purification and Kinetic Characterization of 6-Phosphofructo-1-kinase from the Liver of Gilthead Sea Bream (Sparus Aurata)

Dominica Mediavilla, Isidoro Metón and Isabel V. Baanante*

Departament de Bioquímica i Biologia Molecular, Facultat de Farmàcia, Universitat de Barcelona, Diagonal 643, 08028 Barcelona, Spain

*To whom correspondence should be addressed. Tel: +34-934-02-4521, Fax: +34-934-02-4520, E-mail: baanantevazquez{at}ub.edu

Received March 5, 2008; Accepted April 27, 2008


  Abstract

6-Phosphofructo-1-kinase (PFK) was purified to homogeneity from liver of gilthead sea bream (Sparus aurata) and kinetic properties of the enzyme were determined. The native enzyme had an apparent molecular mass of 510 kDa and was composed of 86 kDa subunits, suggesting homohexameric structure. At pH 7, S. aurata liver PFK (PFKL) showed sigmoidal kinetics for fructose-6-phosphate (fru-6-P) and hyperbolic kinetics for ATP. Fructose-2,6-bisphosphate (fru-2,6-P2) converted saturation curves for fru-6-P to hyperbolic and activated PFKL synergistically with AMP. Fru-2,6-P2 counteracted the inhibition caused by ATP, ADP and citrate. Compared to the S. aurata muscle isozyme, PFKL had lower affinity for fru-6-P, higher cooperativity, hyperbolic kinetics in relation to ATP, increased susceptibility to inhibition by ATP, and was less affected by AMP, ADP and inhibition by 3-phosphoglycerate, phosphoenolpyruvate, 6-phosphogluconate or phosphocreatine. The effect of starvation-refeeding on PFKL expression was studied at the levels of enzyme activity and protein content in the liver of S. aurata. Our findings indicate that short-term recovery of PFKL activity after refeeding previously starved fish, may result from allosteric regulation by fru-2,6-P2, whereas combination of activation by fru-2,6-P2 and increase in protein content may determine the long-term recovery of the enzyme activity.

Key Words: glycolysis, kinetics, liver, 6-phosphofructo-1-kinase, Sparus aurata

Abbreviations: fru-2, 6-P2 fructose-2,6-bisphosphate; fru-6-P, fructose-6-phosphate; PFK, 6-phosphofructo-1-kinase; PFKL, liver 6-phosphofructo-1-kinase; PFKM, muscle 6-phosphofructo-1-kinase; PMSF, phenylmethylsulphonyl fluoride


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