AGGREGATION STATES OF MITOCHONDRIAL MALATE-DEHYDROGENASE

The oligomeric state of fluorescein-labeled mitochondrial malate dehyd rogenase (L-malate NAD(+) oxidoreductase; mMDH; EC 1.1.1.37), as a fun ction of protein concentration, has been examined using steady-state a nd dynamic polarization methodologies. A ''global'' rotational relaxat ion time of 103 +/- 7 ns was found for micromolar concentrations of mM DH-fluorescein, which is consistent with the reported size and shape o f mMDH. Dilution of the mMDH-fluorescein conjugates, prepared using a phosphate buffer protocol, to nanomolar concentrations had no signific ant effect on the rotational relaxation time of the adduct, indicating that the dimer-monomer dissociation constant for mMDH is below 10(-9) M. In contrast to reports in the literature suggesting a pH-dependent dissociation of mMDH, the oligomeric state of this mMDH-fluorescein p reparation remained unchanged between pH 5.0 and 8.0. Application of h ydrostatic pressure up to 2.5 kilobars was ineffective in dissociating the mMDH dimer. However, the mMDH dimer was completely dissociated in 1.5 M guanidinium hydrochloride. Dilution of a mMDH-fluorescein conju gate, prepared using a Tris buffer protocol, did show dissociation, wh ich can be attributed to aggregates present in these preparations. The se results are considered in light of the disparities in the literatur e concerning the properties of the mMDH dimer-monomer equilibrium.