Evaluation of basic mitochondrial functions using rat tissue homogenates

Pecinová, Alena; Drahota, Z; Nůsková, Hana; Pecina, Petr; Houštěk, J

doi:10.1016/j.mito.2011.05.006

Cited by 61 publications

(57 citation statements)

References 28 publications

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“…Measurement of Mitochondrial Oxygen Consumption Rate of Tissue Homogenates-Respiration of intact mitochondria in tissue homogenates was determined as described (55), using freshly harvested kidney tissues, following treatment with AMPK activator and inhibitor as above after homogenization in homogenization buffer with 10 strokes. The protein concentration was determined using the DC protein assay kit (Bio-Rad), and diluted to 0.2 mg/ml with KCl buffer (80 mM KCl, 10 mM Tris-HCl, 3 mM MgCl, 1 mM EDTA, 5 mM potassium phosphate, 2.5 mM malate, 0.5 mg/ml BSA, pH 7.4).…”

Section: Measurement Of Rates Of Oxidation and Reduction-mentioning

confidence: 99%

Phosphorylation of Cytochrome c Threonine 28 Regulates Electron Transport Chain Activity in Kidney

Mahapatra

Varughese

et al. 2017

Journal of Biological Chemistry

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Edited by Dennis R. VoelkerMammalian cytochrome c (Cytc) plays a key role in cellular life and death decisions, functioning as an electron carrier in the electron transport chain and as a trigger of apoptosis when released from the mitochondria. However, its regulation is not well understood. We show that the major fraction of Cytc isolated from kidneys is phosphorylated on Thr 28 , leading to a partial inhibition of respiration in the reaction with cytochrome c oxidase. To further study the effect of Cytc phosphorylation in vitro, we generated T28E phosphomimetic Cytc, revealing superior behavior regarding protein stability and its ability to degrade reactive oxygen species compared with wild-type unphosphorylated Cytc. Introduction of T28E phosphomimetic Cytc into Cytc knock-out cells shows that intact cell respiration, mitochondrial membrane potential (⌬⌿ m ), and ROS levels are reduced compared with wild type. As we show by high resolution crystallography of wild-type and T28E Cytc in combination with molecular dynamics simulations, Thr 28 is located at a central position near the heme crevice, the most flexible epitope of the protein apart from the N and C termini. Finally, in silico prediction and our experimental data suggest that AMP kinase, which phosphorylates Cytc on Thr 28 in vitro and colocalizes with Cytc to the mitochondrial intermembrane space in the kidney, is the most likely candidate to phosphorylate Thr 28 in vivo. We conclude that Cytc phosphorylation is mediated in a tissuespecific manner and leads to regulation of electron transport chain flux via "controlled respiration," preventing ⌬⌿ m hyperpolarization, a known cause of ROS and trigger of apoptosis.

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Section: Measurement Of Rates Of Oxidation and Reduction-mentioning

confidence: 99%

Phosphorylation of Cytochrome c Threonine 28 Regulates Electron Transport Chain Activity in Kidney

Mahapatra

Varughese

et al. 2017

Journal of Biological Chemistry

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“…50-100mg of the native sample was homogenized by a technique which perserves mitochondria in their cytosolic context [3]. Oxygen consumption was measured by high-resolution respirometry (Oxygraph-2Km Oroboros) under two occasions: 1. homogenate enriched with substrates was sequentially treated with an ATPase inhibitor (oligomycine), an uncoupler (FCCP) and KCN.…”

Section: Methodsmentioning

confidence: 99%

The role of mitochondrial dysfunction in the pathophysiology of ICU-acquired weakness

et al. 2014

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“…Mitochondria from rat tissues (heart, liver, kidney, brain, skeletal Abbreviations: ANT, ADP/ATP translocase; PiC, inorganic phosphate carrier; BAT, brown adipose tissue; DDM, n-dodecyl b-D-maltoside. muscle, brown adipose tissue) were isolated by differential centrifugation [12,13].…”

Section: Animalsmentioning

confidence: 99%

“…The separation of proteins with the tricine SDS-PAGE [20] was performed on 10% polyacrylamide minigels (MiniProtean III, BioRad) [13]. The blue native electrophoresis (BN-PAGE) [21] was performed on 4e13 or 4e8 % gradient gels [13].…”

Section: Electrophoreses and Immunoblottingmentioning

confidence: 99%

Mitochondrial ATP synthasome: Expression and structural interaction of its components

Nůsková

Mráček

Mikulová

et al. 2015

Biochemical and Biophysical Research Communications

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Evaluation of basic mitochondrial functions using rat tissue homogenates

Cited by 61 publications

References 28 publications

Phosphorylation of Cytochrome c Threonine 28 Regulates Electron Transport Chain Activity in Kidney

Phosphorylation of Cytochrome c Threonine 28 Regulates Electron Transport Chain Activity in Kidney

The role of mitochondrial dysfunction in the pathophysiology of ICU-acquired weakness

Mitochondrial ATP synthasome: Expression and structural interaction of its components

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