期刊
ANALYTICAL BIOCHEMISTRY
卷 389, 期 2, 页码 157-164出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ab.2009.03.040
关键词
Pyruvate dehydrogenase; Mitochondria; Dichloroacetate; Dichloroacetic acid; Leigh's syndrome; Reversible phosphorylation; Phospho-peptide; Phospho-antibody
资金
- National Institutes of Health (NIH) [18849, DK054441]
- NIH pharmacology training grant [NIH 2 T32 GM07752-25]
- UCSD Foundation Chrisitini Fund
- Lennox Foundation
- Hailey's Wish Foundation
- Wright Family Foundation
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 El alpha subunit of pyruvate dehydrogenase (PDHE1 alpha). 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 PDHE1 alpha and, therefore, overall metabolism in the disease state as well as in response to a myriad of physiological and pharmacological stimuli. (C) 2009 Elsevier Inc. All rights reserved.
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