title: Subunit dissociation in the allosteric regulation of glycerol kinase from Escherichia coli. 2. Physical evidence.
abstract: The dependence of the molecular weight of glycerol kinase on enzyme concentration and on binding of fructose 1,6-bisphosphate has been examined by velocity sedimentation, gel filtration, and polyacrylamide gel electrophoresis. The sedimentation coefficient and Stokes radius decrease as a consequence of dilution in a manner consistent with dissociation into half-molecules, with limiting values suggesting molecular weights of about 218,000 and 136,000 for the associated and dissociated species, respectively. Fructose 1,6-bisphosphate (5 mM) prevents the decrease in sedimentation coefficient brought about by dilution, suggesting a decrease in the apparent subunit dissociation constant of at least four orders of magnitude. Electrophoretic mobility in polyacrylamide gels increases as a consequence of dilution in the absence, but not in the presence, of fructose 1,6-bisphosphate. Ferguson plots indicate that glycerol kinase has the same molecular weight in the presence of fructose 1,6-bisphosphate as the covalently cross-linked tetramer and is substantially smaller in the absence of fructose 1,6-bisphosphate. These results are consistent with the model of glycerol kinase proposed in the preceding paper of this issue [de Riel, J.K., and Paulus, H. (1978), Biochemistry 17] relating subunit dissociation and ligand binding.
mesh_terms: D000494:Allosteric Regulation; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D004926:Escherichia coli / Q000201:enzymology*; D005635:Fructosediphosphates / Q000494:pharmacology; D005991:Glycerol Kinase / Q000037:antagonists & inhibitors*; D046911:Macromolecular Substances; D008970:Molecular Weight; D010770:Phosphotransferases / Q000037:antagonists & inhibitors*; D011487:Protein Conformation
chemical_list: D005635:Fructosediphosphates; D046911:Macromolecular Substances; D010770:Phosphotransferases; D005991:Glycerol Kinase