Cytosolic phosphofructokinase from spinach leaves : I. Purification, characteristics, and regulation

Cytosolic ATP-dependent phosphofructokinase (PFK) from spinach leaves (Spinacia oleracea L.) was enriched 2600-fold by (NH₄)₂SO₄ fractionation, DEAE anion exchange chromatography, Blue Sepharose CL-6B, and ATP agarose type 3-affinity chromatography. The final preparation had a specific activity of 417 nkat per milligram protein and exhibited four bands between 50 and 70 kilodaltons following denaturing electrophoresis. Only one band of ATP- and fructose 6-phosphate (F-6-P)-dependent, Pistimulated activity was detected following isoelectric focusing PAGE and nondenaturing discontinuous PAGE of the final preparation. Crude extracts contained, in addition to the band observed in the final preparation, a second band that was inhibited by Pi. The latter band is presumably chloroplastic PFK. PFK was stimulated by the anions Pi²⁻, Cl⁻, SO₄²⁻, NO₃⁻, HAsO₄²⁻, and HCO₃⁻ but was not affected by NH₄⁺. Pi and Mg²⁺ changed the response of PFK toward pH and affected the saturation kinetics of F-6-P. In general, activity was highest when Pi was high and (or) Mg²⁺ was low. Phosphoenolpyruvate (PEP), 2-PGA, and PPi, but not 3-PGA, inhibited PFK. Although the inhibition by PEP and 2-PGA was reduced or relieved by Pi, the inhibition by PPi was not affected by Pi. F-2, 6-P₂ had no effect upon the activity of PFK. It is proposed that, in the cytosol of spinach leaves, PFK is likely to be more active during the dark, when cytosolic Pi levels are high, than in the light.