title: Aggregation states of mitochondrial malate dehydrogenase.
abstract: The oligomeric state of fluorescein-labeled mitochondrial malate dehydrogenase (L-malate NAD+ oxidoreductase; mMDH; EC 1.1.1.37), as a function of protein concentration, has been examined using steady-state and dynamic polarization methodologies. A "global" rotational relaxation time of 103 +/- 7 ns was found for micromolar concentrations of mMDH-fluorescein, which is consistent with the reported size and shape of mMDH. Dilution of the mMDH-fluorescein conjugates, prepared using a phosphate buffer protocol, to nanomolar concentrations had no significant 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 preparation remained unchanged between pH 5.0 and 8.0. Application of hydrostatic 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 conjugate, prepared using a Tris buffer protocol, did show dissociation, which can be attributed to aggregates present in these preparations. These results are considered in light of the disparities in the literature concerning the properties of the mMDH dimer-monomer equilibrium.
authors.affiliation: Instituto de Química, Universidad Católica de Valparaíso, Chile.
authors.forename: S A
authors.initials: SA
authors.lastname: Sánchez
authors.forename: T L
authors.initials: TL
authors.lastname: Hazlett
authors.forename: J E
authors.initials: JE
authors.lastname: Brunet
authors.forename: D M
authors.initials: DM
authors.lastname: Jameson
mesh_terms: D000818:Animals; D002850:Chromatography, Gel; D002942:Circular Dichroism; D019281:Dimerization; D005452:Fluoresceins / Q000737:chemistry; D005454:Fluorescence Polarization; D019791:Guanidine / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D008291:Malate Dehydrogenase / Q000737:chemistry*; D008929:Mitochondria, Heart / Q000201:enzymology*; D011487:Protein Conformation; D013552:Swine
chemical_list: D005452:Fluoresceins; D008291:Malate Dehydrogenase; D019791:Guanidine