Rat liver phosphofructokinase: kinetic activity under near-physiological conditions.
Academic Article
Overview
Research
Identity
Additional Document Info
Other
View All
Overview
abstract
Rat liver phosphofructokinase (PFK) has been purified to near homogeneity by the combination and modification of two existing procedures. the kinetic properties of the purified enzyme have been studied under the near-physiological conditions of pH 7.0, 120 mM KC1, and 3 mM MgATP. Assays performed under these conditions are generally nonlinear with time; the shape of the progress curves are influenced by the order of addition of substrates to the reaction mixture. High initial velocities, which decelerate with time, are obtained after initiation with Mg2+, or ATP, whereas accelerating low initial rates occur in assays begun by the addition of fructose 6-phosphate (F6P). the steady-state activity of rat liver PFK, attained after the completion of either type of transient, is characterized by an extremely low affinity and high degree of positive cooperativity toward varying F6P concentration (Km = 6 mM; Hill coefficient > 4). the primary mode of action of the physiological activators (AMP, fructose 1, 6-bisphosphate, and inorganic phosphate) and inhibitors (MgATP, citrate, and H+) studied is to alter the Km for F6P, with little or no effect on Vmax. the simultaneous action of the activators at physiologically optimal concentrations produces a synergistic response which mitigates MgATP inhibition to a large extent. A substantial decrease in MgATP inhibition is also found to be necessary to explain the affinity for F6P that PFK must demonstrate in vivo given the low levels of F6P which are found in the cell. 1980, American Chemical Society. All rights reserved.
