Purification and properties of spinach leaf phosphofructokinase 2/fructose 2,6-bisphosphatase.
Phosphofructokinase 2 was purified from spinach leaves by fractionation with poly(ethylene glycol) and by chromatography on blue Sepharose, anion exchanger Mono-Q and blue Trisacryl. A low-Km fructose-2,6-bisphosphatase copurified with phosphofructokinase 2 and its constitutive subunits could be easily identified by sodium dodecyl sulphate gel electrophoresis thanks to the formation of a [32P]phosphoenzyme intermediate upon short-time incubation in the presence of 1 microM fructose 2,6-[2-32P]bisphosphate. On anion-exchange chromatography, two peaks of phosphofructokinase 2/fructose-2,6-bisphosphatase were resolved. The first one, called L (light), represented about 10% of the phosphofructokinase 2 activity and was characterized by a phosphofructokinase 2/fructose-2,6-bisphosphatase activity ratio close to 1, by an Mr of 132,000 as measured by gel filtration, and by a series of subunits of Mr comprised between 44,000 and 70,000. The second and major peak of phosphofructokinase 2, called H (heavy), had a phosphofructokinase 2/fructose-2,6-bisphosphatase ratio close to 8, an Mr of 390,000 and was made of 90,000-Mr subunits. The H form of phosphofructokinase 2 had a lower Km for fructose 6-phosphate than the L form and a higher Ki for a series of physiological inhibitors. By contrast, the kinetics of fructose-2,6-bisphosphatase was the same for the two forms of the enzyme. Upon incubation in the presence of papain or of a crude spinach leaf extract, the purified H form gave rise to products made of subunits of Mr comprised between 70,000 and 44,000 but also of lower values which maintained their fructose-2,6-bisphosphatase activity. The H and L forms of phosphofructokinase 2/fructose-2,6-bisphosphatase were also detected in crude homogenates of castor bean endosperm and of Jerusalem artichoke tubers.
D000818:Animals; D002845:Chromatography / Q000379:methods; D007700:Kinetics; D008970:Molecular Weight; D010206:Papain; D025481:Phosphofructokinase-2; D010744:Phosphoric Monoester Hydrolases / Q000302:isolation & purification*; D010770:Phosphotransferases / Q000302:isolation & purification*; D010944:Plants / Q000201:enzymology*; D013045:Species Specificity
D010770:Phosphotransferases; D025481:Phosphofructokinase-2; D010744:Phosphoric Monoester Hydrolases; D010206:Papain