Monitoring phosphorylation of the pyruvate dehydrogenase complex

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


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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adolescent
  • Amino Acid Sequence
  • Animals
  • Antibodies / chemistry
  • Antibodies / pharmacology
  • Binding Sites
  • Cell Line
  • Cells, Cultured
  • Child
  • Humans
  • Leigh Disease / enzymology
  • Male
  • Mice
  • Mice, Inbred C57BL
  • Mitochondria / enzymology
  • Molecular Sequence Data
  • Phosphorylation
  • Pyruvate Dehydrogenase Complex / antagonists & inhibitors
  • Pyruvate Dehydrogenase Complex / chemistry*
  • Pyruvate Dehydrogenase Complex / metabolism
  • Rabbits
  • Sequence Alignment
  • Young Adult


  • Antibodies
  • Pyruvate Dehydrogenase Complex