Self-association of rabbit muscle phosphofructokinase: role of subunit interaction in regulation of enzymatic activity.
Phosphofructokinase (PFK) has been reversibly inactivated by oxidized glutathione. The physical properties of this inactivated form were characterized by sedimentation studies. In the presence of saturating amounts of fructose 6-phosphate and the nonhydrolyzable ATP analogue 5'-adenylyl imidodiphosphate, the inactivated PFK sediments as a 13.5S component. Sedimentation equilibrium study identifies it to be a tetramer with a molecular weight of 320 000. Sedimentation velocity studies in buffer alone at both pH 7.00 and pH 8.55 reveal that this inactivated enzyme can still undergo self-association with the same stoichiometry as that of the native enzyme, although the equilibrium constants are in favor of the formation of lower aggregates. The presence of allosteric activators, either ADP or cAMP, enhances the association of the inactivated PFK without changing its stoichiometry, an observation similar to that of the native enzyme. However, quantitatively the enhancement of association is not to the same extent as that of the native enzyme. Conversely, citrate favors the formation of smaller aggregates quantitatively similar to that of the native enzyme. Hence, this study shows that the oxidation state of the protein sulfhydryl groups influences the physical properties of PFK and the modulation of PFK activity is linked to subunit association-dissociation and quaternary structural changes.
D000255:Adenosine Triphosphate / Q000378:metabolism; D000266:Adenylyl Imidodiphosphate / Q000378:metabolism; D000818:Animals; D005636:Fructosephosphates / Q000378:metabolism; D005978:Glutathione / Q000031:analogs & derivatives / Q000378:metabolism; D019803:Glutathione Disulfide; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D009132:Muscles / Q000201:enzymology*; D010732:Phosphofructokinase-1 / Q000378:metabolism*; D011817:Rabbits
D005636:Fructosephosphates; D046911:Macromolecular Substances; D000266:Adenylyl Imidodiphosphate; C027618:fructose-6-phosphate; D000255:Adenosine Triphosphate; D010732:Phosphofructokinase-1; D005978:Glutathione; D019803:Glutathione Disulfide