Phosphofructokinase from white muscle of the rainbow trout, Oncorhynchus mykiss: purification and properties.
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 degrees 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 degrees 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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Department of Chemistry, Carleton University, Ottawa, Ontario, Canada.
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D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002845:Chromatography / Q000379:methods; D002850:Chromatography, Gel; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D007700:Kinetics; D009132:Muscles / Q000201:enzymology; D010732:Phosphofructokinase-1 / Q000737:chemistry / Q000302:isolation & purification*; D012474:Salmon / Q000378:metabolism*; D013696:Temperature
D000596:Amino Acids; D010732:Phosphofructokinase-1