Reversible Dissociation of Tetrameric 7.4 S Rabbit Muscle Glyceraldehyde 3-Phosphate Dehydrogenase into 4.4 S Dimers by Ammonium Sulfate and into 3.2 S Monomers by KCl

Abstract

Abstract At 0°, native tetrameric rabbit muscle glyceraldehyde 3-phosphate dehydrogenase (7.4 S) undergoes a reversible inactivation and dissociation into 4.4 S dimers or 3.2 S monomers, depending upon the species of salt present and upon the protein concentration. These dissociation processes have been studied using sucrose density gradient centrifugation. With 0.10 mg per ml of enzyme in 0.15 m ammonium sulfate, 4.4 S dimers predominate; with 0.05 mg per ml of enzyme in 0.15 m KCl, 3.2 S monomers predominate. At other protein concentrations in the two systems, mixtures are produced. KCl gives greater dissociation at all protein concentrations. It also destabilizes at high salt concentrations (1.0 to 2.5 m). In contrast, destabilization in ammonium sulfate is maximal at 0.15 m; 0.5 and 1.0 m ammonium sulfate stabilize against dissociation essentially completely. The native enzyme in the absence of salts shows very slow, very slight dissociation into dimers. Inactivation and dissociation do not occur at 23° in 0.15 m ammonium sulfate and only slightly in 0.15 m KCl, and then only at very low protein concentrations. Inactivation and dissociation are reversed by warming to 23° and greater than 80% of the activity is recovered. The reversible dissociation and inactivation occur at both pH 7.0 (0.1 m imidazole) and at pH 9.5 (0.2 m glycine). A reducing agent (0.1 m β-mercaptoethanol) has to be present for activity to be recovered. These data prove that this enzyme at 0° exists as an equilibrium mixture which favors association to tetramers at high protein concentrations and dissociation into dimers or monomers or both at low protein concentrations. The dimers and monomers appear to be fairly compact.