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 k 7 ns was found for micromolar concentrations of mMDH-fluorescein, which is consistent with the reported size and shape of mMDH. Dilution of the mMDHfluorescein 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 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.Keywords: dissociation; fluorescence polarization; mitochondrial malate dehydrogenase: time resolved Porcine heart mitochondrial malate dehydrogenase (mMDH) [(S)-malate:NAD+ oxidoreductase, E.C. 1 .I. 1.371 is a dimeric enzyme composed of identical subunits, each with 314 amino acids, which catalyzes the interconversion of L-malate and oxaloacetate utilizing NAD+ as a coenzyme. In 1970s and 1980s, several groups studied the dimer-monomer equilibrium of mMDH using different approaches. Shore and Chakrabarti (1976) reported fluorescence polarization studies on enzyme labeled with either FITC or fluorescamine and, in both cases, reported finding a concentrationdependent dissociation with a dissociation constant (&) equal to 2 X IOu7 M at 23 "C in pH 8.0, 50 mM Tris-acetate buffer. Bleile et al. (1977) and Hodges et al. (1977) reported gel filtration chromatography and sedimentation velocity ultracentrifugation studies indicating dissociation constants for the dimer similar to those reported by Shore and Chakrabarti. Frieden et al. (1978), however, reported enzyme kinetic studies suggesting that mMDH did not undergo dissociation even at 10" M. Jaenicke et al. (1979), using gel filtration chromatography, also concluded that mMDH remained a dimer over the concentration range of 1.67 X IO-' M to 2.9 X M in 0.2 M phosphate buffer, pH 7.6, 20°C. Wood et al. (1978Wood et al. ( , 1981 and Hodges et al. (1977) reported a pH dependence of the dimer/monomer equilibrium, suggesting that pH values below 7 promote dissociation; specifically, a dissociation constant greater than 2 X lop4 M was reported at pH 5.0, while at pH 7.5 this value was given as less than M (...

Amide hydrogen exchange shows that malate dehydrogenase is a folded monomer at pH 5

“…Later studies showed that the dimer dissociates (K D ≈ 200 nM) into monomers at low concentrations of MDH (Shore and Chakrabarti 1976; Bleile et al 1977). However, other groups have reported finding no evidence for dissociation of the monomer (Frieden et al 1978; Jaenicke et al 1979; Sanchez et al 1998). Their results indicate that the dissociation constant for the MDH dimer is less than 3 nM.…”

“…Results from size‐exclusion chromatography (Bleile et al 1977; Wood et al 1978), analytical ultracentrifugation (Hodges et al 1977), and intrinsic fluorescence (Wood et al 1981) indicate that MDH dissociates to monomers at pH 5. However, results of a recent fluorescence polarization study indicate that MDH does not dissociate at pH 5 (Sanchez et al 1998).…”

Identification and characterization of a Hsp70 (DnaK) chaperone system from Meiothermus ruber

“…MDH reactivation was measured at 10-min intervals over a period of for 2 h. MDH activity was measured by the procedure described by Sanchez et al (1998). Identical amounts of E. coli DnaK, DnaJ and GrpE (E. coli chaperones were a kind gift of Dr. H. Pesliakas, Institute of Biotechnology, Vilnius) were employed to induce mitochondrial malate dehydrogenase (MDH) reactivation as a control.…”

Implementation of an experimental and computational tool set to study protein‐mAb interactions

“…Haber and Bennett employed FP to study antibody–antigen interactions to determine the rotational relaxation time of antibody–antigen complexes of different sizes. FP has also been used to study the dynamics of monomer/dimer formation . Royer et al have studied the effect of salt concentration on histone subunit interactions using fluorescence polarization …”

“…FP has also been used to study the dynamics of monomer/dimer formation. [25][26][27] Royer et al have studied the effect of salt concentration on histone subunit interactions using fluorescence polarization. 28,29 Self-interaction chromatography (SIC) is a technique that can provide information about the self-interactions of a given protein, the second virial coefficient (B 22 ) and the effect of mobile phase modifiers on these interactions.…”