Molecular weight of the subunits, oligomeric and associated forms of bovine liver glutamate dehydrogenase

Abstract

The molecular weight of bovine liver glutamate dehydrogenase ( l-glutamate: NAD(P)-oxidoreductase (deaminating) EC 1.4.1.3) has been investigated in 0.2 m-sodium phosphate buffer, pH 7, 10 −3 m-EDTA, by light scattering at 25 °C (at λ = 546 mμ) in the concentration range 0.04 to 8 × 10 −3 g/ml. In the limit of c = 0, the molecular weight M of the enzymically active oligomer equals 316,000; the average value of the molecular weight appears to increase without limit to over 2 × 10 6 at 8 × 10 −3 g/ml. In the presence of 10 −3 m-GTP and 10 −3 m-NADH the enzyme, at finite concentrations, is almost completely dissociated to oligomer units. In the limit of vanishing enzyme concentration M = 310,000 in the presence of these reagents. The average value of the molecular weight of oligomer units was taken as 313,000. The molecular weight of subunits was found to be 53,500, from light scattering experiments in 5.7 m-guanidine hydrochloride, 0.01 m-β-mercaptoethanol. This value is consistent with the value (52,000) which can be derived from equilibrium sedimentation in this medium (Marler & Tanford, 1964) with the use of a partial volume determined by us. It is concluded that six non-covalently bonded subunits form one active oligomer enzyme molecule. From the angular dependence of the scattering of the native enzyme, and with support from known values of the intrinsic viscosity behavior as a function of concentration, it was tentatively concluded that the oligomers associate to form rods, the size of which continuously increases with increase in concentration, over the range studied. Accessory measurements yielded the values 0.177 and 0.130 for the refractive increments ( ∂n ∂c ) μ in phosphate buffer and 5.7 m-guanidine hydrochloride, respectively, and the corresponding values 0.751 and 0.726 for the apparent specific volumes.