Purification of electron transfer flavoprotein from pig liver mitochondria and its application to the diagnosis of deficiencies of acyl-CoA dehydrogenases in human fibroblasts

Bertrand, C.; Dumoulin, R.; Divry, P.; Mathieu, M.; Vianey‐Saban, Christine

doi:10.1016/0009-8981(92)90047-t

Cited by 16 publications

(12 citation statements)

References 15 publications

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“…Mitochondria were purified from calf liver using a combination of differential and Ficoll gradient centrifugation (41,42). All procedures were carried out at 4°C.…”

Section: Preparation Of Mitochondriamentioning

confidence: 99%

Purification and characterization of a mammalian homolog of Escherichia coli MutY mismatch repair protein from calf liver mitochondria

Parker

2000

Nucleic Acids Research

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A protein homologous to the Escherichia coli MutY glycosylase, referred to as mtMYH, has been purified from calf liver mitochondria. SDS-polyacrylamide gel electrophoresis, western blot analysis as well as gel filtration chromatography predicted the molecular mass of the purified calf mtMYH to be 35-40 kDa. Gel mobility shift analysis showed that the purified mtMYH formed specific binding complexes with A/8-oxoG, G/8-oxoG and T/8-oxoG, weakly with C/8-oxoG, but not with A/G and A/C mismatches. The purified mtMYH exhibited DNA glycosylase activity removing adenine mispaired with G, C or 8-oxoG and weakly removing guanine mispaired with 8-oxoG. The mtMYH glycosylase activity was insensitive to high concentrations of NaCl and EDTA. The purified mtMYH cross-reacted with antibodies against both intact MutY and a peptide of human MutY homolog (hMYH). DNA glycosylase activity of mtMYH was inhibited by anti-MutY antibodies but not by antihMYH peptide antibodies. Together with the previously described mitochondrial MutT homolog (MTH1) and 8-oxoG glycosylase (OGG1, a functional MutM homolog), mtMYH can protect mitochondrial DNA from the mutagenic effects of 8-oxoG.

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“…Mitochondria were purified from calf liver using a combination of differential and Ficoll gradient centrifugation (41,42). All procedures were carried out at 4°C.…”

Section: Preparation Of Mitochondriamentioning

confidence: 99%

Purification and characterization of a mammalian homolog of Escherichia coli MutY mismatch repair protein from calf liver mitochondria

Parker

2000

Nucleic Acids Research

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“…All enzyme deficiencies were confirmed by molecular biology except for patient P11 who was only heterozygous for the frequent c.985A>G mutation. Diagnosis was confirmed for this patient by an enzymatic activity assay using ETF as electron acceptor (Bertrand et al 1992). All the identified mutations (Table 1) have been described as deleterious in the literature and/or by predictions software (Alamut ® , Interactive biosoftware).…”

Section: Population Studiedmentioning

confidence: 76%

“…The MCAD residual activity of 8% confirms plasma acylcarnitine and urine organic acid analyses. This method is easier and shorter to achieve than the previous method using ETF (Bertrand et al 1992).…”

Section: Discussionmentioning

confidence: 95%

Development of a Tandem Mass Spectrometry Method for Rapid Measurement of Medium- and Very-Long-Chain Acyl-CoA Dehydrogenase Activity in Fibroblasts

et al. 2016

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Mitochondrial fatty acid oxidation is a vital biochemical process for energy metabolism. Among the known fatty-acid metabolism disorders, very-long-chain acyl-CoA dehydrogenase (VLCAD) deficiency and medium-chain acyl-CoA dehydrogenase (MCAD) deficiency count among the most frequent. Both are potentially very serious diseases as they carry a risk of severe neurological post-crisis sequelae, and even sudden death. Diagnosis relies on plasma acylcarnitine profile analysis and urine organic acid analysis, followed by genetic testing to confirm diagnosis. However, in some cases, it is crucial to run a specific diagnostic assay for enzyme activity, which is generally performed in leukocytes or fibroblasts. The aim of this study was to address this need, first by developing a MCAD and VLCAD enzyme activity-specific diagnostic assay in fibroblasts (by measuring the reaction products, i.e. enoyl-CoA) via a rapid LC-MS/MS-based technique, and then by testing MCAD-deficient patients (n ¼ 6), VLCADdeficient patients (n ¼ 10), and control patients (n ¼ 12). MCAD activity was significantly different in the MCADdeficiency (MCADD) group (mean ¼ 0.07 nmol C8:1 formed/min/mg protein) compared to the control group (mean ¼ 0.36 nmol C8:1 formed/min/mg protein). All MCADD patients showed less than 35% residual MCAD activity. VLCAD activity was significantly decreased in the VLCADD group (mean ¼ 0.06 nmol C16:1 formed/min/ m g p r o t e i n ) c o m p a r e d t o t h e c o n t r o l g r o u p (mean ¼ 0.86 nmol C16:1 formed/min/mg protein, respectively). All VLCADD patients showed less than 35% residual VLCAD activity. This technique allowed also to confirm that a novel ACADVL gene mutation (c.1400T>C) is responsible for a defective VLCAD activity (residual activity at 10%).

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“…Studies of 14C-labelled fatty acid oxidation were performed in intact fibroblasts, using [1-14C] The activity of long-chain (LCAD), medium-chain (MCAD) and n = 20 short-chain (SCAD) acyl-CoA dehydrogenases was determined in fibroblasts using the decrease in fluorescence of the natural electron acceptor ETF with pahnitoyl-CoA, octanoyl-CoA and butyrylCoA as substrates as previously published [2]. All experiments were performed in triplicate The activity of mitochondrial fatty acyl-CoA dehydrogenases was measured in fibroblasts using the ETF reduction assay [2].…”

Section: Methodsmentioning

confidence: 99%

“…All experiments were performed in triplicate The activity of mitochondrial fatty acyl-CoA dehydrogenases was measured in fibroblasts using the ETF reduction assay [2].…”

Section: Methodsmentioning

confidence: 99%

Carnitine palmitoyl transferase I deficiency presenting as a Reye-like syndrome without hypoglycaemia

et al. 1993

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An apparently healthy girl aged 2 years 9 months developed a coma with hepatomegaly within 24 h after an influenza-like infection. Plasma glucose and urinary organic acid profile were normal but plasma and urinary carnitine concentrations were increased. Despite symptomatic therapy, she died 11 days later. Oxidation of [1-14C] palmitic acid in the patient's fibroblasts was severely decreased (13% of controls). Further investigations revealed a deficiency of carnitine palmitoyl transferase I (CPT I) in the patient's fibroblasts (15% of controls) whereas CPT II activity was normal. Only four patients with CPT I deficiency have been reported so far. The subtle clinical and biochemical presentation of this disorder, which may account for the small number of cases diagnosed, is discussed.

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Purification of electron transfer flavoprotein from pig liver mitochondria and its application to the diagnosis of deficiencies of acyl-CoA dehydrogenases in human fibroblasts

Cited by 16 publications

References 15 publications

Purification and characterization of a mammalian homolog of Escherichia coli MutY mismatch repair protein from calf liver mitochondria

Purification and characterization of a mammalian homolog of Escherichia coli MutY mismatch repair protein from calf liver mitochondria

Development of a Tandem Mass Spectrometry Method for Rapid Measurement of Medium- and Very-Long-Chain Acyl-CoA Dehydrogenase Activity in Fibroblasts

Carnitine palmitoyl transferase I deficiency presenting as a Reye-like syndrome without hypoglycaemia

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