Glutamate dehydrogenase : I.Flourescence polarization studies of the self-association. II.Flourescence and circular dichroism studies of the heterotropic interactions Public Deposited

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  • Fluorescence polarization measurements on beef liver glutamate dehydrogenase conjugated with pyrene butyric acid demonstrate that the association of the enzyme is adequately described by a reversible indefinite association with a single equilibrium constant. The data obtained with protein concentrations up to 0.7 mg/ml in 0.05 M potassium phosphate, pH 7. 6, are consistent with a dissociation constant of 0.275 mg/ml for an end to end association. The rotational relaxation time of the beef liver glutamate dehydrogenase monomer (320, 000 g/mole) is 1030 ± 70 nsecs at 20°. The retention of catalytic activity and sensitivity to effectors, ADP and GTP, shows that labeling causes little or no detectable change in the properties of the enzyme. Parallel experiments with dogfish glutamate dehydrogenase confirm the absence of significant association in this enzyme. Together these two cases demonstrate the usefulness of fluorescence polarization in the study of proteins undergoing self-association. The binding of reduced pyridine nucleotides to beef liver glutamate dehydrogenase and dogfish liver glutamate dehydrogenase has been examined by circular dichroism and fluorescence. Beef liver glutamate dehydrogenase binds six moles of NADPH both in the presence and absence of GTP. This is consistent with the hexamer hypothesis for the oligomer. In agreement with previous evidence, there are multiple sites for NADH. In the presence of GTP, 18 sites per hexamer have been fluorometrically titrated and confirmed by sedimentation. Formation of the abortive ternary complex indicates that six of these sites are equivalent to the NADPH binding sites. The coenzyme binding equilibrium of beef liver glutamate dehydrogenase is independent of protein concentration in the range examined (0.2 - 2.0 mg/ml). Dogfish glutamate dehydrogenase, anon- associating form from a primitive vertebrate, is NAD⁺(H) specific but shows the same heterogeneity in the binding of NADH.
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