Abstract
The quaternary structure of an active form of rabbit muscle phosphofructokinase was studied by sedimentation and electron microscopy. Active enzyme centrifugation studies at pH 7.0 and 23 +/- 1 degrees C showed that phosphofructokinase sediments as a single component with a sedimentation coefficient of 12.2 +/- 0.5 S. Identical results were obtained in two assay and three solvent systems. Boundary sedimentation studies of phosphofructokinase in the presence of 1.0 mM fructose 6-phosphate, 0.1 mM adenylyl imidodiphosphate at pH 7.0 and 23 +/- 1 degrees C were performed. The results showed that the sedimentation coefficient of phosphofructokinase remains constant within the range of protein concentration studied and assumes a value of 12.4 S. The molecular weights of the subunit and the 12.4 S component were measured by sedimentation equilibrium yielding values of 83,000 and 330,000 for the monomeric and polymeric species, respectively. It is, therefore, concluded that the active form of phosphofructokinase is indeed the tetrameric species. The structure of the phosphofructokinase tetramer was also studied by electron microscopy of negatively stained specimens. Particles identified as tetramers measured approximately 9 nm in diameter by 14 nm in length. The observed size and shape are consistent with the hydrodynamic measurements. Structural features within the tetramer were interpreted as due to the four individual subunits, each one approximately 4 X 6 X 6 nm in size, arranged with D2 symmetry.
Highlights
The quaternary structure of an active form of rabbit assume different gross conformations in the presence of varmuscle phosphofructokinase was studied by sedimen- ious ligands
The structure of the phosphofructokinase tetramer not sufficient, to determine the structural features was studied by electron microscopy of negatively of an oligomeric enzyme or an enzyme such as phosphofrucstained specimens
A knowledge of the structure anhdydrodynamic properties measurementsinaddition to active enzyme centrifugation of an enzyme molecule under conditions where it is fully active with the goal of characterizing the structure and hydrodyis important for understanding the enzymic reaction at the namic properties of the active form of phosphofructokinase
Summary
Active enzyme centrif- versible association-dissociation, the structural changes obugation studies a t pH 7.0 and 23 & 1 “C showed that served in these studies might be partly or wholly due to the phosphofructokinase sediments as a single component physical constraints of the cross-links or aneffect of chemical with a sedimentation coefficient of 12.2 2 0.5 S. The low concentration of enzyme utilized by this technique imentation coefficient of phosphofructokinase remains makes it possible in theory to correlate the physical state of constantwithintherange of proteinconcentration the enzyme with its kinetic properties. It is possible studied and assumes a value of 12.4 S. 1973; Parr and Hammes, 1975, 1976; Hill and Hammes, 1975; Lad et al, 1973)
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