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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

1
Department of Pharmacology, University of California, San Diego, La Jolla, CA 92093, USA.

Abstract

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.

PMID:
19341700
PMCID:
PMC2713743
DOI:
10.1016/j.ab.2009.03.040
[Indexed for MEDLINE]
Free PMC Article

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