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.

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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