Recent evidence indicates that in as diverse organisms as unicellular eukaryotes, higher plants and prokaryotes, anaerobic glycolysis relies on a pyrophosphate-dependent phosphofructokinase instead of the classical ATP-dependent enzyme. This difference in phosphoryl donor specificity does not necessarily reflect a primitive metabolism, as thought earlier, but could rather be the result of convergent evolution, fostered by the energetic advantage conferred to the cell when glycolysis is the sole source of ATP.

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Phosphofructokinase 2 was purified from spinach leaves by fractionation with poly(ethy1ene glycol) and by chromatography on blue Sepharose, anion exchanger Mono-Q and blue Trisacryl. A low-K, 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 pM 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 M , of 132000 as measured by gel filtration, and by a series of subunits of M , comprised between 44000 and 70000. The second and major peak of phosphofructokinase 2, called H (heavy), had a phosphofructokinase 2/fructose-2,6-bisphosphatase ratio close to 8, an M , of 390000 and was made of 90000-M, subunits. The H form of phosphofructokinase 2 had a lower K,,, 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 M, comprised between 70000 and 44000 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.Fructose 2,6-bisphosphate is a newly discovered regulator of metabolism present in most eukaryotic cells (reviewed in [l -31). It is synthesized from Fru6P and ATP by PFK 2 and is hydrolyzed back to Fru6P and Pi by FBPase 2. In liver and muscle, PFK 2 and FBPase 2 are part of a single bifunctional dimeric protein with an M , close to I10000 [2 -51. Liver and yeast PFK 2 are substrates for cyclic-AMP-dependent protein kinase which causes the inactivation of the enzyme in liver but its activation in yeast [3]. Some kinetic properties of PFK 2 and FBPase 2 present in a partially purified preparation of spinach leaves have also been reported [6-8a] but the enzymes have not been characterized at the molecular level.

Rice (Oryza sativa) seeds were imbibed for 3 days and the seedlings were further incubated for 8 days in the presence of either air or nitrogen. In aerobiosis, the specific activity of pyrophosphate:fructose 6-phosphate 1-phosphotransferase and that of the ATP-dependent phosphofructokinase increased about fourfold. In anaerobiosis, the specific activity of ATP-dependent phosphofructokinase remained stable, whereas that of pyrophosphate:fructose 6-phosphate 1-phosphotransferase increased as much as in the presence of oxygen and there was also a fourfold increase in the concentration of fructose 2,6-bisphosphate, a potent stimulator of that enzyme. These data suggest a preferential involvement of pyrophosphate:fructose 6-phosphate 1-phosphotransferase rather than of ATP-dependent phosphofructokinase in glycolysis during anaerobiosis.