Studies of Glutamate Dehydrogenase

The size and shape of beef liver glutamate dehydrogenase were investigated by X‐ray small‐angle measurements in the range of protein concentration between 1 and 33 mg/ml. It was shown that the cross‐section of the glutamate dehydrogenase particles of varying sizes were independent of the molecular weight of the particles. The radius of gyration of the cross‐section ( R q ) was found to be 30.3 Å and the mass per unit length ( M /1Å) to be 2,340. From the comparison of the experimental scattering curves with theoretical cross‐section curves for elliptical cylinders it follows that the glutamate dehydrogenase molecule has a very isotropic cross‐section. The experimental data lie between the axial ratio 1:1 (circular cross‐section) and 0.8:1. For the circular cross‐section a diameter of 86 Å and a cross‐section area of 5,800 Å 2 is calculated. For an axial ratio of 0.8:1 the long and the short axes are calculated to be 95 Å and 76 Å, respectively, and the cross‐section area is found to be 5,700 Å 2 . Again the cross‐section is independent of protein concentrations and therefore independent of the state of association of glutamate dehydrogenase. This result confirms our earlier conclusion from diffusion, sedimentation and viscosity measurements, that the dissociation of the prolate glutamate dehydrogenase molecule into enzymatically active subunits involves a transverse cleavage. A linear relationship exists between the length and the molecular weight of the glutamate dehydrogenase particles. For a molecular weight of about two millions a length of 800–900 Å was found, for molecular weights of one and one‐half millions the length of the molecules were calculated to 400 Å and 200 Å, respectively. These data are confirmed by comparison of the experimental data with theoretical scattering curves of circular cylinders. The results support the conclusion that the associated enzyme molecule is formed in a stepwise association‐dissociation equilibrium. The scattering curves of glutamate dehydrogenase show a pronounced side maximum at an angle corresponding to a Bragg's value of 56 Å. The comparison with theoretical cross‐section curves of hollow cylinders suggests that glutamate dehydrogenase is loosely built and possesses voids. This suggestion is supported by the difference of the volume of glutamate dehydrogenase per 1 Å length calculated from the mass per 1 Å length (4,200 Å 3 , even if one assumes an average swelling of proteins of 0.33 g H 2 O per 1 g protein) and from the cross‐section area (5,700 to 5,800 Å 3 ).