Crosstalk between Mitochondrial Malate Dehydrogenase and Cytochrome bc1 Complex

Abstract

The interactions between the mitochondrial cytochrome bc 1 complex and matrix-soluble proteins were studied by a precipitation pulldown technique. Purified, detergent-dispersed bc 1 complex was incubated with mitochondrial matrix proteins followed by dialysis in the absence of detergent. The interacting protein(s) was co-precipitated with bc 1 complex upon centrifugation. One of the matrix proteins pulled down by bc 1 complex was identified as mitochondrial malate dehydrogenase (MDH) by matrix-assisted laser desorption ionization time-of-flight mass spectrometry and confirmed by Western blotting with anti-MDH antibody. Using a cross-linking technique, subunits I, II (core I and II), and V of the bc 1 complex were identified as the interacting sites for MDH. Incubating purified MDH with the detergent dispersed bc 1 complex results in an increase of the activities of both the bc 1 complex and MDH. The effect of the bc 1 complex on the activities of MDH is unidirectional (oxaloacetate 3 malate). These results suggest that the novel cross-talk between citric acid cycle enzymes and electron transfer chain complexes might play a regulatory role in mitochondrial bioenergetics.

Effects of putrescine on oxidative stress induced by hydrogen peroxide in Salvinia natans L.

“…These two enzymes are operative in a sequential manner for biosynthesis of proline and have been evident to relate the redox balance under osmotic stress (Thippeswamy et al 2010). Recently, the effects of cadmium and zinc stress on these enzyme activities have been studied and revealed the regulatory property of proline in homeostasis of ions (Wang et al 2010). However, in the present experiment, the activity of γ-GK and its down regulation under H 2 O 2 were recorded.…”

“…0.1 mL of extract was added to reaction buffer containing 0.1 mM of adenosine triphosphate (ATP), incubated at 37°C for 30 min subsequently added with 2 mL of stop solution. γ-GK activity was measured spectrophotometrically at 535 nm and expressed as unit (U) defined as µg of γ-glutamyl hydroxamate formed min −1 mg −1 protein (Wang et al 2010). Similar extraction buffer was used for γ-GPR (EC:1.2.1.41) activity under identical condition except 0.1 M cysteine was used to stabilize the protein.…”

Identification of the Components of a Glycolytic Enzyme Metabolon on the Human Red Blood Cell Membrane

“…Questions remained, however, regarding which population of band 3 might be interacting with the GEs and whether these interactions were specific or simply driven by nonselective interactions (41). To explore the nature of these interactions further, we used a trifunctional photocross-linking reagent (sulfo-SBED) that has been employed successfully by many laboratories, including our own (42), to identify nearest neighbor pro- teins in complex macromolecular structures and organelles (43)(44)(45). The photocross-linking reagent has a length of 1.4 nm and generates a nitrene upon photoactivation, enabling insertion into any proximal peptide (Pierce).…”

Inhibition of Akt Phosphorylation Diminishes Mitochondrial Biogenesis Regulators, Tricarboxylic Acid Cycle Activity and Exacerbates Recognition Memory Deficit in Rat Model of Alzheimer’s Disease

“…Measurement of Malate Dehydrogenase (MDH) Activity MDH activity was measured using NADH following the conversion of oxaloacetate to malate as described by Wang et al (2010). By the addition of 0.1 ml of freshly isolated sample to assay mixture composed of 5.6 mM oxaloacetate in Tris-HCl (56 mM, pH 7.5) and 10 ll of NADH solution containing 0.5 mg NADH in 100 ll of TrisHCl (50 mM, pH 9), the reaction was initiated.…”