Subunit dissociation of mitochondrial malate dehydrogenase.

Fluorescence polarization studies of porcine mitochondrial malate dehydrogenase labeled with fluorescein isothiocyanate or fluorescamine indicated a concentration-dependent dissociation of the dimeric molecule with a KD OF 2 X 10(7) N at pH 8.0. These results were confirmed by the concentration dependence of the stability of the enzyme at elevated temperatures and the creation of hybrid molecules with fluorescein and Rhodamine B labeled subunits, in which energy transfer was observed. The binding of NADH resulted in a small shift of the subunit dissociation curve toward monomer, demonstrating that monomer has twice the affinity for reduced coenzyme. NAD+ binding prevented dissociation of the dimer, even at concentrations below 10(-8) N. These results indicate that binding of reduced or oxidized coenzymes results in different conformation changes, which are transferred to the subunit interface.