Quantitative analysis of mixed association between different protein molecules. Physico-chemical and enzymatic properties of rat-liver glutamate dehydrogenase.
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 light-scattering measurements. Its value (1.3 x 10(-4) M(-1)) 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 subs-rates 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.
D000818:Animals; D002417:Cattle; D002942:Circular Dichroism; D005969:Glutamate Dehydrogenase / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008027:Light; D008099:Liver / Q000201:enzymology*; D046911:Macromolecular Substances; D008433:Mathematics; D011485:Protein Binding; D011487:Protein Conformation; D051381:Rats; D012542:Scattering, Radiation; D013045:Species Specificity
D046911:Macromolecular Substances; D005969:Glutamate Dehydrogenase