Urea and methylamine effects on rabbit muscle phosphofructokinase. Catalytic stability and aggregation state as a function of pH and temperature.

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The effects of urea and several methylamine solutes on the catalytic stability and aggregation properties of rabbit muscle phosphofructokinase were assessed at physiologically realistic concentrations of the solutes under several pH and temperature regimes. The loss of catalytic activity observed under conditions of pH-induced cold lability was significantly reduced in the presence of trimethylamine-N-oxide, N-trimethylglycine and N-methylglycine (order of decreasing effectiveness). The concentration-dependent methylamine stabilization of the enzyme, seen with as little as 50 mM trimethylamine-N-oxide, was accompanied by increased aggregation of the enzyme to molecular weights greater than the tetramer (polytetramer) as solute concentration was raised to 400 mM. At pH 6.5-6.7 and 25 degrees C, concentrations of urea greater than 25 mM promoted a time-dependent inactivation of the enzyme which was enhanced at lower temperatures. The urea sensitivity of the enzyme exhibited with 0.8 M urea for 1 h at pH 8.0 did not result in measurable inactivation. The fluorescence emission wavelength maximum of the enzyme was shifted to longer wavelengths and the fluorescence intensity was increased as pH was lowered to 7.0, suggesting the occurrence of a protein conformation change as specific amino acid residues of the tetramer became protonated. Measurements of enzyme light scattering indicated that perturbation by urea was correlated with tetramer dissociation, which was irreversible by dialysis at 25 degrees C. The urea and methylamine influences on phosphofructokinase activity and structure were not counteracting. The synergistic interactions among pH, temperature, and solutes observed with phosphofructokinase are compared to effects on other associating-dissociating protein systems in order to evaluate possible mechanisms of action of these low molecular weight solutes.

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Journal of Biological Chemistry

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