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Metabolism and Enzymology

Cytosolic Phosphofructokinase from Spinach Leaves ¹

II. Affinity for Mg²⁺ and Nucleoside Phosphates

Botanisches Institut der Westfälischen Wilhelms-Universität, Schloßgarten 3, D-4400 Münster, Federal Republic of Germany

Cytosolic ATP-phosphofructokinase (PFK) from spinach leaves (Spinacia oleracea L.) was inhibited by submillimolar concentrations of free Mg²⁺. The free Mg²⁺ concentration required for 50% inhibition of PFK activity was 0.22 millimolar. Inhibition by free Mg²⁺ was independent of the MgATP^2– concentration. Inorganic phosphate (Pi) reduces the inhibition of PFK activity by Mg²⁺. 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 Mg²⁺ 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 Ca²⁺. The inhibition by Ca²⁺ depends upon the total Mg²⁺ concentration. Fifty percent inhibition of PFK activity required 22 and 32 micromolar Ca²⁺ at 0.1 and 0.2 millimolar Mg²⁺, respectively. At physiological concentrations of about 0.5 millimolar free Mg²⁺, Ca²⁺ 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 Mg²⁺ and Pi.

¹ This work was supported by grants from the Deutsche Forschungsgemeinschaft.