Recently we reported' that the glutamic dehydrogenase (GDH) reaction, catalyzed by the crystalline beef liver enzyme or by mitochondrial extracts of rat liver, was inhibited by steroids and diethylstilbestrol (DES), and that this inhibition could be reversed by ADP. Because of the physiological importance of this enzyme, we have extended our investigations in an attempt to determine the mechanism of the inhibition.In the work to be presented, kinetic experiments suggested a rather complicated interaction between the enzyme and the steroids.2 In view of the interesting findings of Frieden3' 4 that DPNH and various inhibitors of GDH could dissociate the protein into enzymically inactive subunits, we were interested in whether the steroid inhibitors could induce similar changes.Our data show that various steroid hormones can, indeed, produce alterations in the physical state of the enzyme, as reflected in sedimentation characteristics.Materials and Methods.-Crystalline beef liver GDH, suspended in Na2SO4 solution, and the nucleotides were obtained from the Sigma Chemical Company. Steroids and DES were obtained from various commercial sources.The rate of oxidation of glutamate by either TPN or DPN at room temperature was followed spectrophotometrically by observing the change in optical density at 340 mA, between 15 and 30 sec, as described by Frieden.3 The reduction of a-ketoglutarate was likewise observed spectrophotometrically and the initial rate was again calculated from the change between 15 and 30 sec after the start of the reaction, although it followed zero order kinetics for a longer period.Steroids were dissolved in 50% propylene glycol and the solutions were maintained at 1000 before addition to the reaction mixtures in order to prevent precipitation. In the kinetic experiments, the final concentration of propylene glycol in the reaction mixture was 2% by volume, which did not affect the rate in either direction. Before ultracentrifugation, the crystalline GDH was dissolved in 0.05 M tris (hydroxymethyl) aminomethane-HCl buffer, pH 8, containing 5 X 10-4 M ethylenediamine tetraacetic acid (EDTA) and the solution was maintained at 00. Steroids were added in 50 per cent propylene glycol as described above, and appropriate controls were run with propylene glycol alone.Sedimentation experiments were performed in the Spinco Model E analytical ultracentrifuge,5 equipped with automatic temperature control, at a rotor speed of 59,780 rpm. Photographs were taken at 4-min intervals, and sedimentation coefficients, calculated from 4 exposures, were correct to 200, assuming that the viscosity of the solvent (containing propylene glycol) varied with temperature in the same way as that of water. This assumption seemed justified by the observation that S20,W of the enzyme was not changed significantly when the propylene glycol concentration was doubled. 1483
