The theoretical and experimental analysis of a reversible association-dissociation equilibrium between different proteins (mixed association) is described. The experiments were performed with glutamate dehydrogenases from beef and rat liver. These enzymes are different, especially with respect to their association behavior.The association constant of rat liver glutamate dehydrogenase has been determined by lightscattering measurements. Its value (1.3 x M-') is much lower than that of the beef liver enzyme, but the difference in the free association energy is only 30 %. Association between these two enzymes is observed, also employing light-scattering experiments. Theoretical curves for mixed associating systems have been calculated and by comparison with these curves the mixed association constant could be determined. Since the free association energy of the mixed association is very near to the arithmetic mean between the values for the pure enzymes, the association interactions appear to be additive. The model of an open association with a virial coefficient is also true for the rat enzyme and the mixed association. The ultracentrifuge data are also explained by the same model and yield a similar value for the mixed association constant. Differences in the enzyme kinetics are small, but a somewhat reduced lifetime of the ternary complexes with the coenzymes and with substrates or GTP can be concluded for the rat liver enzyme. The circular dichroism measurements indicate no significant difference in the dissociation constants of the nucleotides, but the different amplitudes of the ellipticity indicate small differences in the electrical environment of the active center.In protein chemistry it is a well known phenomenon that protein molecules can undergo a reversible association reaction. Such an association can occur between identical or different protein molecules, e.g. between enzymes and membrane proteins. The quantitative analysis of such interacting systems has been performed until now only for those including identical components and not for those with different components. In this paper we describe the theoretical and experimental treatment of systems consisting of different components.Several mammalian glutamate dehydrogenases exhibit a reversible association equilibrium between an enzymatically active subunit (unimer') and asThis work has been described partly in a preliminary report [l]. For the previous paper in this series see [2].Enzyme. Glutamate dehydrogenase or L-glutamate : NAD(P) oxidoreductase (deaminating) (EC 1.4.1.3).' The polypeptide chain of glutamate dehydrogenase has a molecular weight of 56000. Six polypeptide chains form a Eur. J. Biochem. 52 (1975) sociated particles [3]. Rat liver glutamate dehydrogenase, however, was first described to be unable to undergo this association although other properties, including peptide maps, are close to those of the beef liver enzyme [4,5]. Contrary to these experiments we demonstrated in a previous paper by sedimentation analysis a weak associa...
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