Studies of Glutamate Dehydrogenase

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Abstract
The investigation of the association‐dissociation equilibrium of beef liver glutamate dehydrogenase by light‐scattering measurements over a wide range of protein concentration (0.02 to 50 mg/ml) has shown that this equilibrium can be described as an open equilibrium (Mi+n Mh⇋ Mi+h, i and h≥ 1, M = oligomer) with identical equilibrium constants for all steps and without any limit. The dependence of the apparent weight‐average molecular weight on the protein concentration reaches a maximum at about 9 mg/ml. The decrease of the molecular weight at higher protein concentrations must be attributed to effects of nonideality (as expressed in relation to a first approximation by the second virial coefficient). At 20°C and in M/15 sodium phosphate buffer the equilibrium constant was found to be 9.0 × 105 M−1 (corresponding to a free energy change of 7.8 kcal/mol for each consecutive step in the association reaction) and the second virial coefficient to be 9 nmol × 1 × g−2. The calculation of the dependence of the reduced viscosity on the protein concentration based on these results conforms with the experimental data. However, it was found that the length of the oligomer obtained from light‐scattering measurements was dependent on the wavelength. The reason for this phenomenon is not quite obvious.Low pH and low ionic strength promote the association of the oligomers. It was found that the sedimentation coefficient at pH 6.2 and an ionic strength of 0.04 is 31 S (34 S in the presence of 4.4 mM NAD+) compared to 27 S at an ionic strength of 0.115. The pH dependence of the sedimentation coefficient at low ionic strength indicates the participation of a group in the association with a pK near 7, possibly an imidazole group. At high ionic strength the sedimentation coefficient is almost pH independent.From the results it may be concluded that, in the interaction between the oligomers at low ionic strength, ionic effects are involved that are shielded at higher ionic strength.GTP and NADH cause the dissociation into the oligomer characterized by an s020,w value of 13.0 ± 0.3. The dissociation is complete in the presence of 20 mM GTP and NADH. The result that the s020,w values of the oligomer are the same in the absence and in the presence of effectors, GTP/NADH, conforms to our X‐ray small‐angle measurements.