Purification and kinetic properties of 6-phosphofructo-1-kinase from gilthead sea bream muscle.
The kinetic properties of 6-phosphofructo-1-kinase (PFK) from skeletal muscle (PFKM) of gilthead sea bream (Sparus aurata) were studied, after 10,900-fold purification to homogeneity. The native enzyme had an apparent molecular mass of 662 kDa and is composed of 81 kDa subunits, suggesting a homooctameric structure. At physiological pH, S. aurata PFKM exhibited sigmoidal kinetics for the substrates, fructose-6-phosphate (fru-6-P) and ATP. Fructose-2,6-bisphosphate (fru-2,6-P(2)) converted the saturation curves for fru-6-P to hyperbolic, activated PFKM synergistically with other positive effectors of the enzyme such as AMP and ADP, and counteracted ATP and citrate inhibition. The fish enzyme showed differences regarding other animal PFKs: it is active as a homooctamer, and fru-2,6-P(2) and pH affected affinity for ATP. By monitoring incorporation of (32)P from ATP, we show that fish PFKM is a substrate for the cAMP-dependent protein kinase. The mechanism involved in PFKM activation by phosphorylation contrasts with previous observations in other species: it increased V(max) and did not affect affinity for fru-6-P. Unlike the mammalian muscle enzyme, our findings support that phosphorylation of PFKM may exert a major role during starvation in fish muscle.
Mediavilla
Dominica
D
Departament de Bioquímica i Biologia Molecular, Facultat de Farmàcia, Universitat de Barcelona, Diagonal 643, 08028 Barcelona, Spain.
Metón
Isidoro
I
Baanante
Isabel V
IV
D000255:Adenosine Triphosphate / Q000378:metabolism; D000494:Allosteric Regulation; D000818:Animals; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D029941:Fish Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D005635:Fructosediphosphates / Q000378:metabolism; D005636:Fructosephosphates / Q000378:metabolism; D005947:Glucose / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D018482:Muscle, Skeletal / Q000201:enzymology*; D010732:Phosphofructokinase-1 / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D010766:Phosphorylation; D011487:Protein Conformation; D021122:Protein Subunits; D021541:Sea Bream / Q000378:metabolism*; D013379:Substrate Specificity
D029941:Fish Proteins; D005635:Fructosediphosphates; D005636:Fructosephosphates; D021122:Protein Subunits; C027618:fructose-6-phosphate; C027652:fructose 2,6-diphosphate; D000255:Adenosine Triphosphate; D010732:Phosphofructokinase-1; D017868:Cyclic AMP-Dependent Protein Kinases; D005947:Glucose