Send to

Choose Destination
Anal Biochem. 2009 Jun 15;389(2):157-64. doi: 10.1016/j.ab.2009.03.040. Epub 2009 Mar 31.

Monitoring phosphorylation of the pyruvate dehydrogenase complex.

Author information

Department of Pharmacology, University of California, San Diego, La Jolla, CA 92093, USA.


The pyruvate dehydrogenase multienzyme complex (PDC) is a key regulatory point in cellular metabolism linking glycolysis to the citric acid cycle and lipogenesis. Reversible phosphorylation of the pyruvate dehydrogenase enzyme is a critical regulatory mechanism and an important point for monitoring metabolic activity. To directly determine the regulation of the PDC by phosphorylation, we developed a complete set of phospho-antibodies against the three known phosphorylation sites on the E1 alpha subunit of pyruvate dehydrogenase (PDHE1alpha). We demonstrate phospho-site specificity of each antibody in a variety of cultured cells and tissue extracts. In addition, we show sensitivity of these antibodies to PDH activity using the pyruvate dehydrogenase kinase-specific inhibitor dichloroacetate. We go on to use these antibodies to assess PDH phosphorylation in a patient suffering from Leigh's syndrome. Finally, we observe changes in individual phosphorylation states following a small molecule screen, demonstrating that these reagents should be useful for monitoring phosphorylation of PDHE1alpha and, therefore, overall metabolism in the disease state as well as in response to a myriad of physiological and pharmacological stimuli.

[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for Elsevier Science Icon for PubMed Central
Loading ...
Support Center