The basic objective of this investigation is to elucidate the thermodynamics and forces responsible for the intermolecular interaction of phosphofructokinase, a system that has been extensively investigated. Yet there is no information on the modes of self association; the stoichiometry of the polymeric form and the forces involved in subunit interaction have yet to be elucidated. However, results from preliminary sedimentation experiments in our laboratory indicate that the quaternary structure of PFK is altered in the presence of enzyme assay mixture. It is, therefore, proposed to investigate the thermodynamic parameters which govern the self association of PFK subunits by sedimentation and light scattering. The effects of temperature, pH and pressure on the formation of such aggregates will be followed. Such studies should yield information on the mode of association, the stoichiometry and hydrodynamic properties of the aggregates formed and the forces involved. It is further proposed to identify the enzymic active oligomers of PFK from rabbit tissues and individual isozymes from ascites hepatoma cells whose PFK exhibits comparatively high activity and different electrophoretic mobility. The proposed reacting enzyme sedimentation technique, as demonstrated by our preliminary data, could yield information on the hydrodynamic properties of the enzyme-substrate complex enabling a direct correlation between physical properties and kinetic data. The effects of metabolites on the equilibrium between active and inactive forms of the enzyme will be studied with concomitant monitoring of the conformation of the enzyme by fluorescence energy transfer measurements.
NIH ExPorter
NIH ExPorter