The relation of the pH and concentration-dependent dissociation of porcine heart mitochondrial malate dehydrogenase

Wood, David; Hodges, C T; Harrison, John

doi:10.1016/0006-291x(78)90874-4

Cited by 22 publications

(12 citation statements)

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“…This conclusion is also supported by size‐exclusion chromatography, which showed that MDH at pH 7 has an apparent molecular mass of 60 kD, whereas MDH equilibrated at pH 5 has an apparent molecular mass of 30 kD (Chen and Smith 2000). Similar results have been reported previously (Bleile et al 1977; Wood et al 1978). Finding that MDH dissociates to monomers at pH 5 is also consistent with a study in which His 46, which is located in the subunit interface, was mutated to Leu (Steffan and McAlister‐Henn 1991).…”

Section: Resultssupporting

confidence: 93%

“…Additional studies have shown that dissociation of MDH depends on pH. 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).…”

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confidence: 99%

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

Chen

Smith

2001

Protein Science

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Although there is general agreement that native mitochondrial malate dehydrogenase (MDH) exists as a dimer at pH 7, its aggregation state at pH 5 is less certain. The present amide hydrogen exchange study was performed to determine whether MDH remains a dimer at pH 5. To detect pH-induced changes in solvent accessibility, MDH was exposed to D 2 O at pH 5 or 7, then fragmented with pepsin into peptides that were analyzed by mass spectrometry. Even after adjustments for the effect of pH on the intrinsic rate of hydrogen exchange, large increases in deuterium levels were found at pH 5 only in peptic fragments derived from the subunit binding surface of MDH. In parallel experiments, elevated deuterium levels were also found in the same regions of MDH monomer trapped inside a mutant form of the chaperonin GroEL. This selective increase in hydrogen exchange rates, which was attributed to increased solvent accessibility of these regions, provides new evidence that MDH is a monomer at pH 5. Keywords: Malate dehydrogenase; amide hydrogen exchange; mass spectrometryResults of an early study of malate dehydrogenase (MDH) showed that this enzyme is a homodimer (Devenyi et al. 1966). 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. Additional studies have shown that dissociation of MDH depends on pH. 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).Resolution of these opposing views of the quaternary structure of MDH is important for our understanding of the mechanism of its function and for studies in which it has been used as a model to study the role of subunit interactions in enzymology (Wood et al. 1981;Steffan and McAlister-Henn 1991;Chen and Smith 2000). To help resolve this controversy, hydrogen exchange and mass spectrometry were used in the present study to investigate the quaternary structure of MDH at pH 5.0. X-ray crystallography shows that the two subunits in MDH have extensive contacts with each other (Gleason et al. 1994), which indicates that hydrogen exchange may be a highly specific method for distinguishing between monomeric and dimeric forms of MDH (Hvidt and Nielsen 1966;Englander and Kallenbach 1984;Mandell et al. 1998). In this report, we present evidence that MDH is a folded monomer at pH 5. Results and DiscussionThe aggregation state of MDH at pH 5 was studied by comparing the hydrogen exchange kinetics in intact MDH at pH 5 and 7. Hydrogen ...

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Section: Resultssupporting

confidence: 93%

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confidence: 99%

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

Chen

Smith

2001

Protein Science

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“…The pH dependence of the dimer/monomer transition of mMDH has been addressed by Harrison and his collaborators (Bleile et al, 1977;Wood et al, 1978Wood et al, , 1981. Their findings, in part, have motivated other groups to investigate the subunit interface of mMDH using site-directed mutagenesis.…”

Section: Discussionmentioning

confidence: 99%

“…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 re-mained 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 (Wood et al, 1978). A more recent report (McKay & Jameson, 1991) on mMDH using fluorescein-labeled protein and fluorescencepolarization methods indicated that highly active enzyme remained dimeric at M , and suggested that the disparity in the literature may have been due to the variable specific activities of the different preparations studied.…”

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Aggregation states of mitochondrial malate dehydrogenase

et al. 1998

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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 (...

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“…All proteins were initially at the same molar concentrations in the monolayer subphase and the experihiental points show the surfacepressure change induced after the protein injection at various initial monolayer surface pressures. Under the subphase conditions and in the absence of added coenzyme the malate dehydrogenase will be at least 80% dissociated (Wood et al, 1978). The pressure increases resulting from malate dehydrogenase absorption are considerably greater than those caused by the other two proteins at all surface pressures and at both air/water and oil/water interfaces.…”

Section: Resultsmentioning

confidence: 96%

Hydrophobic interaction between the monomer of mitochondrial malate dehydrogenase and phospholipid membranes

Webster

Freeman²,

Ohki³

1980

Biochemical Journal

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Porcine mitochondrial malate dehydrogenase (EC 1.1.1.37) dissociates into subunits on dilution. The enzyme monomer caused large increases in the surface pressure of monolayers of 1:1 phosphatidylserine/phosphatidylcholine at air/water and oil/water interfaces. The monomer increased the permeability of phospholipid vesicles to 22Na+. Both effects were significantly greater than the corresponding effects of ribonuclease A, cytochrome c and the dimeric form of malate dehydrogenase. Changes in the circular-dichroism spectra of the enzyme indicated that conformational changes may be associated with dimer formation or when monomer interacts with lysophosphatidyl-choline. Similar interactions to those described may occur in situ when mitochondrial malate dehydrogenase is transported to the mitochondrial matrix from its site of synthesis on cytosolic ribosomes.

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The relation of the pH and concentration-dependent dissociation of porcine heart mitochondrial malate dehydrogenase

Cited by 22 publications

References 10 publications

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

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

Aggregation states of mitochondrial malate dehydrogenase

Hydrophobic interaction between the monomer of mitochondrial malate dehydrogenase and phospholipid membranes

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