title: Purification of phosphofructokinase using transition-state analogue affinity chromatography.
abstract: A novel purification of phosphofructokinase has been achieved in a two step process using ion-exchange affinity chromatography and a transition-state analogue affinity column matrix. The procedure can be performed in one day, and gives a 25% yield of the starting material. The transition-state analogue chromatography is carried out using an ADP-agarose column in the presence of fructose 6-phosphate, magnesium ions and nitrate ions. In the presence of nitrate ion plus substrate, phosphofructokinase binds immobilized ADP while other proteins pass through the column. Previous studies with creatine kinase have shown that the nitrate ion mimics the planar phosphate in the transition state resulting in a complex which is stable under the relatively high ionic strength of the column buffer. This permits the elution of phosphofructokinase in a single peak of high specific activity. This column typically results in a 20-30 fold increase in specific activity with only a small loss of activity.
authors.affiliation: Institute of Biochemistry, Carleton University, Ottawa, Ontario, Canada.
authors.forename: S P
authors.initials: SP
authors.lastname: Brooks
authors.forename: K B
authors.initials: KB
authors.lastname: Storey
mesh_terms: D000818:Animals; D002846:Chromatography, Affinity; D010732:Phosphofructokinase-1 / Q000302:isolation & purification*; D014426:Turtles
chemical_list: D010732:Phosphofructokinase-1