Rat liver phosphofructokinase: use of fluorescence polarization to study aggregation at low protein concentration.
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The aggregation properties of rat liver phosphofructokinase (PFK) have been studied at low protein concentration by measuring the depolarization of fluorescence of PFK covalently labeled with pyrenebutyric acid. When PFK is labeled at a stoichiometry of between 0.3 and 1 pyrene per 3.2 105 daltons, kinetic integrity of the enzyme is maintained as determined by maximal specific activity and the cooperative response to fructose 6-phosphate (F6P) concentration at high MgATP concentration at pH 7. the fluorescence characteristics of the labeled enzyme (PB-PFK) are appropriate for the interpretation of polarization changes as reflecting changes in the average size, or rotational relaxation time, of the protein population. Consistent with the results of gel filtration and active enzyme ultracentrifugation experiments, the polarization data indicate that liver PFK is quite capable of forming very high molecular weight aggregates (50 S) at a protein concentration of 10, g/mL. In isotonic pH 7 buffer containing excess Mg2+, the dissociation constant of the high Mt aggregates is lower in the presence of saturating F6P than it is in the presence of saturating MgATP. In the absence of either substrate, PB-PFK dissociates past the tetrameric form of the enzyme with a concomitant loss in enzymatic activity. Subsequent addition of MgATP promotes tetramer formation much more than does the addition of F6P. These data are interpreted as indicating that the monomer-dimer population has a lower affinity for F6P than for MgATP whereas the high Mt aggregate population has a higher affinity for F6P than for MgATP. 1980, American Chemical Society. All rights reserved.
