Modified native electrophoresis protocol for the solubilization and separation of mitochondrial protein complexes

Cunha, Elizabeth Sousa da; Domingues, Cleyton C.; Paula, Eneida de

doi:10.1016/j.ab.2011.06.044

Cited by 5 publications

(3 citation statements)

References 6 publications

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“…CI is present in a lower molecular mass complex in CHO mitochondria (750 -800 kDa, Fig. 12A) than in rat/ bovine heart mitochondria (ϳ1000 kDa) (77) but is similar in size to rat muscle CI (78). The strong in-gel activity of this band (I 1 in Fig.…”

Section: Cell Size and Mitochondrial Morphology Of Mtpe-deficientmentioning

confidence: 98%

Phosphatidylethanolamine Deficiency in Mammalian Mitochondria Impairs Oxidative Phosphorylation and Alters Mitochondrial Morphology

Tasseva

Bai

Davidescu

et al. 2013

Journal of Biological Chemistry

274

258

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Background:The contribution of phosphatidylethanolamine (PE) to mammalian mitochondrial function was unknown. Results: A decrease in mitochondrial PE impairs cell growth, respiratory capacity, and ATP production and profoundly alters mitochondrial morphology. Conclusion: Mitochondrial PE is required for normal morphology and function of mammalian mitochondria. Significance: Modest reduction of mitochondrial PE might contribute to mitochondrial dysfunction in some disease states.

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Section: Cell Size and Mitochondrial Morphology Of Mtpe-deficientmentioning

confidence: 98%

Phosphatidylethanolamine Deficiency in Mammalian Mitochondria Impairs Oxidative Phosphorylation and Alters Mitochondrial Morphology

Tasseva

Bai

Davidescu

et al. 2013

Journal of Biological Chemistry

274

258

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“…The separation of the protein complexes was performed with 200 nM of each protein in non-denaturing conditions according to the modified protocol [22]. For the second dimension, proteins were denatured in the gel strip after separation by BN-PAGE and subsequently subjected to SDS-PAGE, as previously described [23].…”

Section: Blue Native Polyacrylamide Gel Electrophoresismentioning

confidence: 99%

In Vitro Characterization of a Nuclear Receptor-like Domain of the Xylanase Regulator 1 from Trichoderma reesei

Mello-de-Sousa

Gorsche

Jovanović

et al. 2022

JoF

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Engineering transcription factors is an interesting research target gaining increasing attention, such as in the case of industrially used organisms. With respect to sustainability, biomass-degrading saprophytic fungi, such as Trichoderma reesei, are promising industrial work horses because they exhibit a high secretory capacity of native and heterologously expressed enzymes and compounds. A single-point mutation in the main transactivator of xylanase and cellulase expressions in T. reesei Xyr1 led to a strongly deregulated and enhanced xylanase expression. Circular dichroism spectroscopy revealed a change in secondary structure caused by this mutation. According to electrophoretic mobility shift assays and determination of the equilibrium-binding constants, the DNA-binding affinity of the mutated Xyr1 was considerably reduced compared to the wild-type Xyr1. Both techniques were also used to investigate the allosteric response to carbohydrates (D-glucose-6-phosphate, D-xylose, and sophorose) signalling the repression or induction of Xyr1 target genes. The mutated Xyr1 no longer exhibited a conformational change in response to these carbohydrates, indicating that the observed deregulation is not a simple matter of a change in DNA-binding of the transactivator. Altogether, we postulate that the part of Xyr1 where the mutation is located functions as a nuclear receptor-like domain that mediates carbohydrate signals and modulates the Xyr1 transactivating activity.

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“…6 Most techniques involve measuring enzyme activity and using ETC protein immunoblotting to detect protein abundance 7,8 and their utility in the clinical setting in patients with disease is not clear. 9,10…”

mentioning

confidence: 99%

Hybrid gel electrophoresis using skin fibroblasts to aid in diagnosing mitochondrial disease

Newell

Khan²,

Sinasac³

et al. 2019

Neurol Genet

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ObjectiveWe developed a novel, hybrid method combining both blue-native (BN-PAGE) and clear-native (CN-PAGE) polyacrylamide gel electrophoresis, termed BCN-PAGE, to perform in-gel activity stains on the mitochondrial electron transport chain (ETC) complexes in skin fibroblasts.MethodsFour patients aged 46–65 years were seen in the Metabolic Clinic at Alberta Children's Hospital and investigated for mitochondrial disease and had BN-PAGE or CN-PAGE on skeletal muscle that showed incomplete assembly of complex V (CV) in each patient. Long-range PCR performed on muscle-extracted DNA identified 4 unique mitochondrial DNA (mtDNA) deletions spanning the ATP6 gene of CV. We developed a BCN-PAGE method in skin fibroblasts taken from the patients at the same time and compared the findings with those in skeletal muscle.ResultsIn all 4 cases, BCN-PAGE in skin fibroblasts confirmed the abnormal CV activity found from muscle biopsy, suggesting that the mtDNA deletions involving ATP6 were most likely germline mutations that are associated with a clinical phenotype of mitochondrial disease.ConclusionsThe BCN-PAGE method in skin fibroblasts has a potential to be a less-invasive tool compared with muscle biopsy to screen patients for abnormalities in CV and other mitochondrial ETC complexes.

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Modified native electrophoresis protocol for the solubilization and separation of mitochondrial protein complexes

Cited by 5 publications

References 6 publications

Phosphatidylethanolamine Deficiency in Mammalian Mitochondria Impairs Oxidative Phosphorylation and Alters Mitochondrial Morphology

Phosphatidylethanolamine Deficiency in Mammalian Mitochondria Impairs Oxidative Phosphorylation and Alters Mitochondrial Morphology

In Vitro Characterization of a Nuclear Receptor-like Domain of the Xylanase Regulator 1 from Trichoderma reesei

Hybrid gel electrophoresis using skin fibroblasts to aid in diagnosing mitochondrial disease

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