Phosphofructokinase was purified and characterized from the white skeletal muscle of rainbow trout Oncorhynchus mykiss. Purification involved three steps: ion-exchange chromatography on hydroxyapatite and affinity chromatography on phosphocellulose and ATP-agarose. A final specific activity of 75 units per mg of protein at 22°C and pH 7.2 with 40% recovery was obtained. The purified enzyme gave a single band on SDS-PAGE with a subunit molecular mass of 76.5±0.6 kDa. Based on gel filtration analysis, the active form of the enzyme was found to be composed of six identical subunits. A high isoelectric point (7.1) was found for this enzyme. Arrhenius plots of the enzyme activity showed a sharp transition at 15-16°C. The pH optimum of the enzyme was 8.0-8.5 at physiological level of ATP and positive modulators shifted the optimum to lower pH values. Amino-acid analysis revealed a lower content of the aromatic residues Phe, Tyr and Trp and higher level of Ser residue than in the rabbit muscle enzyme.

Characterization, Phosphofructokinase, Purification, Rainbow trout
Biochimica et Biophysica Acta (BBA)/Protein Structure and Molecular
Department of Biology

Su, Y. (Yanjing), & Storey, K. (1992). Phosphofructokinase from white muscle of the rainbow trout, Oncorhynchus mykiss: purification and properties. Biochimica et Biophysica Acta (BBA)/Protein Structure and Molecular, 1160(3), 301–308. doi:10.1016/0167-4838(92)90092-R