Cytosolic Phosphofructokinase from Spinach Leaves : II. Affinity for Mg and Nucleoside Phosphates

Cytosolic ATP-phosphofructokinase (PFK) from spinach leaves (Spinacia oleracea L.) was inhibited by submillimolar concentrations of free Mg(2+). The free Mg(2+) concentration required for 50% inhibition of PFK activity was 0.22 millimolar. Inhibition by free Mg(2+) was independent of the MgATP(2-) concentration. Inorganic phosphate (Pi) reduces the inhibition of PFK activity by Mg(2+). Free ATP (ATP(4-)) also inhibits PFK activity. For free ATP the inhibition of PFK activity was dependent on the MgATP(2-) concentration. Fifty percent inhibition of PFK activity requires 1.2 and 3.7 millimolar free ATP at 0.1 and 0.5 millimolar MgATP(2-), respectively. It was proposed that free ATP competes for the MgATP(2-) binding site, whereas free Mg(2+) does not. Pi diminished the inhibitory effect of free ATP on PFK activity. Free ATP and Pi had substantial effects on the MgATP(2-) requirement of cytosolic PFK. For half-maximum saturation of PFK activity 3 and 76 micromolar MgATP(2-) was required at 0.007 and 0.8 millimolar free ATP in the absence of Pi. At 5 and 25 millimolar Pi, half-maximum saturation was achieved at 9 and 14 micromolar MgATP(2-). PFK activity was inhibited by Ca(2+). The inhibition by Ca(2+) depends upon the total Mg(2+) concentration. Fifty percent inhibition of PFK activity required 22 and 32 micromolar Ca(2+) at 0.1 and 0.2 millimolar Mg(2+), respectively. At physiological concentrations of about 0.5 millimolar free Mg(2+), Ca(2+) would have little effect on cytosolic PFK activity from spinach leaves. PFK is not absolutely specific for the nucleoside 5'-triphosphate substrate. Besides MgATP(2-), MgUTP(2-), MgCTP(2-), and MgGTP(2-) could be used as a substrate. All four free nucleotides inhibit PFK activity. The physiological consequences of the regulatory properties of cytosolic PFK from spinach leaves will be discussed. A model will be introduced, in an attempt to describe the complex interaction of PFK with substrates and the effectors Mg(2+) and Pi.