Blue Native Polyacrylamide Gel Electrophoresis: A Powerful Tool in Diagnosis of Oxidative Phosphorylation Defects

Coster, Rudy Van; Smet, Joél; George, Edith; Meırleır, Linda De; Seneca, Sara; Hove, Johan Van; Sébire, Guillaume; Verhelst, Hélène; Bleecker, Jan De; Vlem, Bruno Van; Verloo, Patrick; Leroy, Jules

doi:10.1203/00006450-200111000-00020

Cited by 123 publications

(91 citation statements)

References 12 publications

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“…Solubilized mitochondria were resuspended in sample buffer (0.5% Coomassie brilliant blue G-250 [Serva], 50 mM 6-aminocaproic acid, 10 mM BisTris-HCl [pH 7.0], and 0.1 mM PMSF), and 300 g protein was separated by electrophoresis with a continuous 5% to 13% gradient blue native polyacrylamide gel electrophoresis (BN-PAGE) gel. For the catalytic staining reaction of complex V, the BN-PAGE gel was incubated in 34 mM Tris, 270 mM glycine, 14 mM MgSO 4 , 0.2% Pb(NO 3 ) 2 , and 8 mM ATP, pH 7.8, overnight at room temperature (49). For immunoblotting, proteins on the BN-PAGE gel were transferred onto a polyvinylidene difluoride membrane and blotted using standard procedures.…”

Section: Methodsmentioning

confidence: 99%

Mammalian Polynucleotide Phosphorylase Is an Intermembrane Space RNase That Maintains Mitochondrial Homeostasis

Chen¹,

Rainey²,

Balatoni³

et al. 2006

Molecular and Cellular Biology

133

150

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We recently identified polynucleotide phosphorylase (PNPase) as a potential binding partner for the TCL1 oncoprotein. Mammalian PNPase exhibits exoribonuclease and poly(A) polymerase activities, and PNPase overexpression inhibits cell growth, induces apoptosis, and stimulates proinflammatory cytokine production. A physiologic connection for these anticancer effects and overexpression is difficult to reconcile with the presumed mitochondrial matrix localization for endogenous PNPase, prompting this study. Here we show that basal and interferon-␤-induced PNPase was efficiently imported into energized mitochondria with coupled processing of the N-terminal targeting sequence. Once imported, PNPase localized to the intermembrane space (IMS) as a peripheral membrane protein in a multimeric complex. Apoptotic stimuli caused PNPase mobilization following cytochrome c release, which supported an IMS localization and provided a potential route for interactions with cytosolic TCL1. Consistent with its IMS localization, PNPase knockdown with RNA interference did not affect mitochondrial RNA levels. However, PNPase reduction impaired mitochondrial electrochemical membrane potential, decreased respiratory chain activity, and was correlated with altered mitochondrial morphology. This resulted in F o F 1 -ATP synthase instability, impaired ATP generation, lactate accumulation, and AMP kinase phosphorylation with reduced cell proliferation. Combined, the data demonstrate an unexpected IMS localization and a key role for PNPase in maintaining mitochondrial homeostasis.

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Section: Methodsmentioning

confidence: 99%

Mammalian Polynucleotide Phosphorylase Is an Intermembrane Space RNase That Maintains Mitochondrial Homeostasis

Chen¹,

Rainey²,

Balatoni³

et al. 2006

Molecular and Cellular Biology

133

150

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“…Diagnostic workup of the cultured skin fibroblasts consisted of spectrophotometric analysis of the activities of complex II (24), III (25), and IV (26) and activity staining of all complexes after separation by blue native-polyacrylamide gel electrophoresis (27) and immunocytochemical staining (28) ( Table 4). On the basis of the results obtained in skeletal muscle and other tissues, subjects were subdivided into patients with isolated OXPHOS defects and those with combined complex I and IV deficiency.…”

Section: Articlesmentioning

confidence: 99%

Fluorescence imaging of mitochondria in cultured skin fibroblasts: a useful method for the detection of oxidative phosphorylation defects

et al. 2012

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Background: Protons are pumped from the mitochondrial matrix via oxidative phosphorylation (OXPhOs) into the intermembrane space, creating an electric membrane potential (ΔΨ) that is used for adenosine triphosphate (aTP) production. Defects in one or more of the OXPhOs complexes are associated with a variety of clinical symptoms, often making it difficult to pinpoint the causal mutation. Methods: In this article, a microscopic method for the quantitative evaluation of ΔΨ in cultured skin fibroblasts is described. The method using 5,5′,6,6′-tetraethylbenzimidazolyl-carbocyanine iodide (Jc-1) fluorescence staining was tested in a selection of OXPhOs-deficient cell lines. results: a significant reduction of ΔΨ was found in the cell lines of patients with either an isolated defect in complex I, II, or IV or a combined defect (complex I + complex IV). ΔΨ was not reduced in the fibroblasts of two patients with severe complex V deficiency. addition of the complex I inhibitor rotenone induced a significant reduction of ΔΨ and perinuclear relocalization of the mitochondria. In cells with a heteroplasmic mitochondrial DNa (mtDNa) defect, a more heterogeneous reduction of ΔΨ was detected. conclusion: Our data show that imaging of ΔΨ in cultured skin fibroblasts is a useful method for the evaluation of OXPhOs functioning in cultured cell lines.t he majority of cellular adenosine triphosphate (ATP) is supplied in the mitochondria through oxidative phosphorylation (OXPHOS). The OXPHOS system consists of five multiprotein complexes embedded within the inner mitochondrial membrane: complex I (reduced nicotinamide adenine dinucleotide: ubiquinone oxidoreductase), complex II (succinate: ubiquinone oxidoreductase), complex III (ubiquinol: cytochrome c oxidoreductase), complex IV (cytochrome c oxidase), and complex V (ATP synthase). Complexes I, III, and IV pump protons, donated by reduced nicotinamide adenine dinucleotide, ubiquinone, and cytochrome c, respectively, into the mitochondrial intermembrane space. The proton gradient that develops between the mitochondrial matrix and the intermembrane space generates an electrochemical membrane potential (ΔΨ), which is the driving force for the conversion of ADP and inorganic phosphate into ATP (1).The estimated incidence of OXPHOS defects in humans is ~1 in 5,000 live births. Isolated complex I deficiency (2) and the mitochondrial DNA (mtDNA) depletion syndromes (3) are the most commonly recognized mitochondrial disorders. Defects in OXPHOS are associated with a broad spectrum of symptoms and syndromes, ranging from mild myopathy to severe multisystem disorders. OXPHOS defects are complex due to the dual origin of genes involved and the specific nature of the mitochondrial genome. The mtDNA encodes 13 structural proteins of the complexes I, III, IV, and V; two rRNAs; and 22 transfer RNAs (tRNAs) necessary for intramitochondrial protein translation. The mitochondrial genome is polyploid, with multiple copies of mtDNA present within each individual mitochondrion. Two different populatio...

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“…Blue native polyacrylamide gel electrophoresis followed by in-gel activity staining Blue native polyacrylamide gel electrophoresis (BN-PAGE) and in-gel activity staining were performed as has been described previously (Van Coster et al 2001). Briefly, mitochondria were prepared from the patient together with a control muscle specimen.…”

Section: Enzyme Assaysmentioning

confidence: 99%

Aberrant synthesis of ATP synthase resulting from a novel deletion in mitochondrial DNA in an African patient with progressive external ophthalmoplegia

Westhuizen

Smet

Levanets

et al. 2010

J of Inher Metab Disea

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Blue Native Polyacrylamide Gel Electrophoresis: A Powerful Tool in Diagnosis of Oxidative Phosphorylation Defects

Cited by 123 publications

References 12 publications

Mammalian Polynucleotide Phosphorylase Is an Intermembrane Space RNase That Maintains Mitochondrial Homeostasis

Mammalian Polynucleotide Phosphorylase Is an Intermembrane Space RNase That Maintains Mitochondrial Homeostasis

Fluorescence imaging of mitochondria in cultured skin fibroblasts: a useful method for the detection of oxidative phosphorylation defects

Aberrant synthesis of ATP synthase resulting from a novel deletion in mitochondrial DNA in an African patient with progressive external ophthalmoplegia

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