Optimized Spectrophotometric Assay for the Completely Activated Pyruvate Dehydrogenase Complex in Fibroblasts

Schwab, Marina A.; Kölker, Stefan; Heuvel, Lambert P. van den; Sauer, Sven W.; Wolf, Nicole I.; Rating, D.; Hoffmann, Georg F.; Smeitink, Jan A.M.; Okun, Jürgen G.

doi:10.1373/clinchem.2004.033852

Cited by 46 publications

(28 citation statements)

References 37 publications

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“…PDC activity was assayed with previously described methods (Jeoung et al, 2006; Schwab et al, 2005). SDH activity was assayed by previously published methods (Nadanaciva et al, 2007).…”

Section: Methodsmentioning

confidence: 99%

SIRT5-Mediated Lysine Desuccinylation Impacts Diverse Metabolic Pathways

et al. 2013

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SUMMARY Protein function is regulated by diverse posttranslational modifications. The mitochondrial sirtuin SIRT5 removes malonyl and succinyl moieties from target lysines. The spectrum of protein substrates subject to these modifications is unknown. We report systematic profiling of the mammalian succinylome, identifying 2,565 succinylation sites on 779 proteins. Most of these do not overlap with acetylation sites, suggesting differential regulation of succinylation and acetylation. Our analysis reveals potential impacts of lysine succinylation on enzymes involved in mitochondrial metabolism; e.g., amino acid degradation, the tricarboxylic acid cycle (TCA) cycle, and fatty acid metabolism. Lysine succinylation is also present on cytosolic and nuclear proteins; indeed, we show that a substantial fraction of SIRT5 is extra-mitochondrial. SIRT5 represses biochemical activity of, and cellular respiration through, two protein complexes identified in our analysis, pyruvate dehydrogenase complex and succinate dehydrogenase. Our data reveal widespread roles for lysine succinylation in regulating metabolism and potentially other cellular functions.

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“…PDC activity was assayed with previously described methods (Jeoung et al, 2006; Schwab et al, 2005). SDH activity was assayed by previously published methods (Nadanaciva et al, 2007).…”

Section: Methodsmentioning

confidence: 99%

SIRT5-Mediated Lysine Desuccinylation Impacts Diverse Metabolic Pathways

et al. 2013

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“…Activities of PDC and KDC were monitored at 340 nm, 30°C, in the presence of 0.1% Triton X-100, as described previously [23] except that components of INT-coupling system and D,L-carnitine were omitted from the assay.…”

Section: Methodsmentioning

confidence: 99%

Compromised Mitochondrial Fatty Acid Synthesis in Transgenic Mice Results in Defective Protein Lipoylation and Energy Disequilibrium

Smith¹,

Witkowski²,

Moghul³

et al. 2012

PLoS ONE

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A mouse model with compromised mitochondrial fatty acid synthesis has been engineered in order to assess the role of this pathway in mitochondrial function and overall health. Reduction in the expression of mitochondrial malonyl CoA-acyl carrier protein transacylase, a key enzyme in the pathway encoded by the nuclear Mcat gene, was achieved to varying extents in all examined tissues employing tamoxifen-inducible Cre-lox technology. Although affected mice consumed more food than control animals, they failed to gain weight, were less physically active, suffered from loss of white adipose tissue, reduced muscle strength, kyphosis, alopecia, hypothermia and shortened lifespan. The Mcat-deficient phenotype is attributed primarily to reduced synthesis, in several tissues, of the octanoyl precursors required for the posttranslational lipoylation of pyruvate and α-ketoglutarate dehydrogenase complexes, resulting in diminished capacity of the citric acid cycle and disruption of energy metabolism. The presence of an alternative lipoylation pathway that utilizes exogenous free lipoate appears restricted to liver and alone is insufficient for preservation of normal energy metabolism. Thus, de novo synthesis of precursors for the protein lipoylation pathway plays a vital role in maintenance of mitochondrial function and overall vigor.

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“…Pyruvate dehydrogenase (PDH) activity was assayed according to conditions previously described by Schwab et al (2005) except that MTT was used as electron acceptor. Additionally, extracts were prepared in extraction buffer without DTT.…”

Section: Direct Assaysmentioning

confidence: 99%

Measurement of key metabolic enzyme activities in mammalian cells using rapid and sensitive microplate‐based assays

Janke

Genzel

Wahl

et al. 2010

Biotech & Bioengineering

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Sensitive microplate-based assays to determine low levels of key enzyme activities in mammalian cells are presented. The enzyme platform consists of four cycling assays to measure the activity of 28 enzymes involved in central carbon and glutamine metabolism. The sensitivity limit of all cycling assays was between 0.025 and 0.4 nmol product. For the detection of glutaminase activity, a new glutamate cycle system involving the enzymes glutamate dehydrogenase and aspartate transaminase was established. The relative standard deviation of the method was found to be 1.7% with a limit of detection of 8.2 pmol and a limit of quantitation of 24.8 pmol. Hence, cell extracts could be highly diluted to reduce interferences caused by other components in the extract, which in addition minimized underestimates or overestimates of actual enzyme activities. Since substrate concentrations could be maintained at a nearly constant level throughout the assay product accumulation during the reaction was low, which minimized product inhibition. As an example, the enzyme platform was used to investigate maximum enzyme activities of stationary-phase MDCK cells grown in serum-containing GMEM medium as typically used in influenza vaccine production.

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Optimized Spectrophotometric Assay for the Completely Activated Pyruvate Dehydrogenase Complex in Fibroblasts

Cited by 46 publications

References 37 publications

SIRT5-Mediated Lysine Desuccinylation Impacts Diverse Metabolic Pathways

SIRT5-Mediated Lysine Desuccinylation Impacts Diverse Metabolic Pathways

Compromised Mitochondrial Fatty Acid Synthesis in Transgenic Mice Results in Defective Protein Lipoylation and Energy Disequilibrium

Measurement of key metabolic enzyme activities in mammalian cells using rapid and sensitive microplate‐based assays

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