Histochemical staining and quantification of dihydrolipoamide dehydrogenase diaphorase activity using blue native PAGE

Yan, Liang; Yang, Shao Hua; Shu, Hongjun; Prókai, László; Forster, Michael J.

doi:10.1002/elps.200600574

Cited by 60 publications

(70 citation statements)

References 49 publications

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“…In T. brucei, dihydrolipoyl dehydrogenase, one of the four subunits of OGDC is shared between OGDC and the pyruvate dehydrogenase complex (16). Its activity was detected using the same approach in mammalian mitochondria, implying that this assay is not specific for complex I activity (49). Furthermore, using a nongradient BNE method, both complex I and dihydrolipoyl dehydrogenase protein were detected simultaneously on the same gel (50), suggesting that the gel-based method used to separate mt respiratory complexes could be critical.…”

Section: Discussionmentioning

confidence: 99%

Trypanosoma brucei Mitochondrial Respiratome: Composition and Organization in Procyclic Form

Acestor

Zı́ková

Dalley

et al. 2011

Molecular & Cellular Proteomics

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The mitochondrial respiratory chain is comprised of four different protein complexes (I-IV), which are responsible for electron transport and generation of proton gradient in the mitochondrial intermembrane space. This proton gradient is then used by F o F 1 -ATP synthase (complex V) to produce ATP by oxidative phosphorylation. In this study, the respiratory complexes I, II, and III were affinity purified from Trypanosoma brucei procyclic form cells and their composition was determined by mass spectrometry. The results along with those that we previously reported for complexes IV and V showed that the respiratome of Trypanosoma is divergent because many of its proteins are unique to this group of organisms. The studies also identified two mitochondrial subunit proteins of respiratory complex IV that are encoded by edited RNAs. Proteomics data from analyses of complexes purified using numerous tagged component proteins in each of the five complexes were used to generate the first predicted protein-protein interaction network of the Trypanosoma brucei respiratory chain. These results provide the first comprehensive insight into the unique composition of the respiratory complexes in Trypanosoma brucei, an early diverged eukaryotic pathogen.

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Section: Discussionmentioning

confidence: 99%

Trypanosoma brucei Mitochondrial Respiratome: Composition and Organization in Procyclic Form

Acestor

Zı́ková

Dalley

et al. 2011

Molecular & Cellular Proteomics

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“…In vitro studies showed that the enzyme is adversely affected by nitrosylation (Yan et al, 2007) suggesting that this might be the case also in vivo. While no decline in dihydrolipoamide dehydrogenase protein levels is observed in normal aging of the brain, a compromised activity was seen in neurodegeneration including Alzheimer’s disease (Gibson et al, 2000).…”

Section: Discussionmentioning

confidence: 99%

Impaired mitochondrial respiration and protein nitration in the rat hippocampus after acute inhalation of combustion smoke

Lee

Reed

Greeley

et al. 2009

Toxicology and Applied Pharmacology

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Survivors of massive inhalation of combustion smoke endure critical injuries, including lasting neurological complications. We have previously reported that acute inhalation of combustion smoke disrupts the nitric oxide homeostasis in the rat brain. In this study, we extend our findings and report that exposure to smoke induces protein nitration in the rat hippocampus and that mitochondrial proteins are a sensitive nitration target in this setting. Mitochondria have central roles in energy metabolism and cellular signaling and are critical to proper cell function. Here, analyses of the mitochondrial proteome showed elevated protein nitration in the course of a 24-h recovery following exposure to smoke. Mass spectrometry identification of several significantly nitrated mitochondrial proteins revealed diverse functions and involvement in central aspects of mitochondrial physiology. The nitrated proteins include the ubiquitous mitochondrial creatine kinase, F1-ATP synthase α subunit, dihydrolipoamide dehydrogenase (E3), succinate dehydrogenase Fp subunit, and voltage-dependent anion channel (VDAC1) protein. Furthermore, acute exposure to combustion smoke significantly compromised the respiratory capacity of hippocampal mitochondria. Interestingly, elevated protein nitration and reduced mitochondrial respiration in the hippocampus persisted beyond the time required for restoration of normal oxygen and carboxyhemoglobin blood levels after the cessation of exposure to smoke. Thus, the data indicate that timing of the different smoke inhalation-induced effects varies among tissues. Taken together, our findings suggest that nitration of essential mitochondrial proteins may contribute to the reduction in mitochondrial respiratory capacity and underlie, in part, the brain pathophysiology after acute inhalation of combustion smoke.

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“…Complexes containing dihydrolipoamide dehydrogenase can also be detected on native gels using NADH and nitroblue tetrazolium as substrates (67). Indeed, immunoblotting of the 4 to 16% native gel with antidihydrolipoamide dehydrogenase (Tb11.01.8470) MAb 17 revealed a band at ϳ500 kDa, the same size as the NADH dehydrogenase activity band (Fig.…”

Section: Respiratory Complex I In Bloodstream T Bruceimentioning

confidence: 92%

Enigmatic Presence of Mitochondrial Complex I in Trypanosoma brucei Bloodstream Forms

et al. 2012

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The presence of mitochondrial respiratory complex I in the pathogenic bloodstream stages of Trypanosoma brucei has been vigorously debated: increased expression of mitochondrially encoded functional complex I mRNAs is countered by low levels of enzymatic activity that show marginal inhibition by the specific inhibitor rotenone. We now show that epitope-tagged versions of multiple complex I subunits assemble into ␣ and ␤ subcomplexes in the bloodstream stage and that these subcomplexes require the mitochondrial genome for their assembly. Despite the presence of these large (740-and 855-kDa) multisubunit complexes, the electron transport activity of complex I is not essential under experimental conditions since null mutants of two core genes (NUBM and NUKM) showed no growth defect in vitro or in mouse infection. Furthermore, the null mutants showed no decrease in NADH:ubiquinone oxidoreductase activity, suggesting that the observed activity is not contributed by complex I. This work conclusively shows that despite the synthesis and assembly of subunit proteins, the enzymatic function of the largest respiratory complex is neither significant nor important in the bloodstream stage. This situation appears to be in striking contrast to that for the other respiratory complexes in this parasite, where physical presence in a life-cycle stage always indicates functional significance.

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Histochemical staining and quantification of dihydrolipoamide dehydrogenase diaphorase activity using blue native PAGE

Cited by 60 publications

References 49 publications

Trypanosoma brucei Mitochondrial Respiratome: Composition and Organization in Procyclic Form

Trypanosoma brucei Mitochondrial Respiratome: Composition and Organization in Procyclic Form

Impaired mitochondrial respiration and protein nitration in the rat hippocampus after acute inhalation of combustion smoke

Enigmatic Presence of Mitochondrial Complex I in Trypanosoma brucei Bloodstream Forms

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