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Structure, regulation and analysis of this key regulatory site in cellular metabolism.
Pyruvate Dehydrogenase complex (PDH) connects the citric acid cycle and subsquent oxidative phosphorylation to the glycolysis, gluconeogenesis and lipid and amino acid metabolism pathways.
PDH catalyzes the oxidative decarboxylation of pyruvate to produce acetyl coenzyme A (acetyl-CoA), NADH and CO2. PDH facilitates the use of carbohydrate to meet energy demands: when carbohydrate stores are depleted in mammals, PDH activity is downregulated to limit the use of glucose by oxidative phosphorylation. Fatty acids or ketone bodies are then used as an energy source in tissues such as heart and skeletal muscle.
PDH is a 9.5 MDa complex consisting of multiple copies of three enzymes: pyruvate dehydrogenase (E1), dihydrolipoamide transacetylase (E2) and dihydrolipoamide dehydrogenase (E3). An additional structural subunit, the E2/E3 binding protein, is necessary to support the interactions between the E2 and E3 subunits.
PDH activity is inhibited by reversible phosphorylation of the E1-alpha subunit at Ser232, Ser293 and Ser300. The phosphorylation of these sites is catalyzed by isoforms of PDH kinases (PDK1 - 4). Dephosphorylation to restore PDH activity is catalyzed by PDH phosphatases (PDP1 and PDP2). Both kinases and phosphatases are differentially expressed in tissues, and their transcription is controlled by a variety of cellular stress events such as starvation, low oxygen or high concentration of NADH, acetyl-CoA or calcium.
Dysregulated use of glucose and fatty acids as energy sources is often observed in metabolic syndromes such as diabetes and obesity. Additionally, cancer cells often switch from oxidative phosphorylation to glycolysis for ATP production. The central role of PDH in these activities has led to a renewed interest in understanding PDH activity and regulation as a potential therapeutic tool.
The approach you choose to study PDH activity depends on experimental conditions and the experimental question you are trying to answer.
Quantification of endogenous and phosphorylated PDH activity in cell and tissue extracts.
The key to measure endogenous PDH activity is to maintain the in vivo state of phosphorylation. Our extraction and immunocapture buffer have been formulated to inhibit specific and non-specific kinases and phosphatases to prevent unwanted PDH modifications during the sample preparation.
Once the samples have been prepared, the active enzyme can be immunocaptured and assayed in solid surfaces using a 96-well microplate format or dipstick assay format. This approach is simpler, faster and safer than the classical method of using [14C]pyruvate and measuring enzyme-catalyzed release of [14C]CO2.
PDH activity can also be analyzed ex vivo following following PDH immunocapture and removal of endogenous kinases and phosphatases. Samples are then phosphorylated with recombinant PDK or dephosphorylated with recombinant PDP to determine (a) residual activity of the fully-phosphorylated PDH, (b) maximum activity of the fully dephosphorylated enzyme and (c) endogenous unmodified PDH activity.
Quantification of total and phosphorylated PDH in live cells or cell and tissue extracts
The key regulatory subunit of PDH is the E1-alpha subunit (PDH1), modified by kinases at three serine phosphorylation sites to decrease activity.
Quantification of total PDH E1-alpha and modified phospho-serines (Ser232, Ser293 and Ser300) can be easily analyzed using sandwich ELISA assays. This approeach measures the levels of protein and phosphorylation, whether they are endogenous or as a result of drug treatment.
In-Cell ELISA (ICE) assays can be used to measure protein levels of post-translational modifications in cultured live cells. The benefit of the ICE method is that rapid fixation of cells in a 96- or 384-well plate in situ stabilizes enzyme phosphorylation and eliminates any changes that may occur during sample preparation.
Analysis of PDH subunits in live cells or cell and tissue extracts by western blot, flow cytometry or immunocytochemistry
Defects in the PDH complex are an important cause of lactic acidosis and cause Leigh's disease in children. The majority of cases of PDH deficiency are due to mutations in the X-linked E1-alpha subunit, despite mutations in the other subunits have also been reported.
Our antibodies have been tested for target specificity by a combination of immunoprecipitation, western blot, immunocytochemistry (ICC) and ELISA assays.