Immunocapture and microplate-based activity measurement of mammalian pyruvate dehydrogenase complex.
Altered pyruvate dehydrogenase (PDH) functioning occurs in primary PDH deficiencies and in diabetes, starvation, sepsis, and possibly Alzheimer's disease. Currently, the activity of the enzyme complex is difficult to measure in a rapid high-throughput format. Here we describe the use of a monoclonal antibody raised against the E2 subunit to immunocapture the intact PDH complex still active when bound to 96-well plates. Enzyme turnover was measured by following NADH production spectrophotometrically or by a fluorescence assay on mitochondrial protein preparations in the range of 0.4 to 5.0 micro g per well. Activity is sensitive to known PDH inhibitors and remains regulated by phosphorylation and dephosphorylation after immunopurification because of the presence of bound PDH kinase(s) and phosphatase(s). It is shown that the immunocapture assay can be used to detect PDH deficiency in cell extracts of cultured fibroblasts from patients, making it useful in patient screens, as well as in the high-throughput format for discovery of new modulators of PDH functioning.
Lib
Margarita
M
Institute of Molecular Biology, University of Oregon, Eugene, Orgon 97403-1229, USA.
Rodriguez-Mari
Adriana
A
Marusich
Michael F
MF
Capaldi
Roderick A
RA
D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D018053:Arsenites / Q000494:pharmacology; D015153:Blotting, Western; D002417:Cattle; D002478:Cells, Cultured; D005347:Fibroblasts / Q000201:enzymology; D006801:Humans; D007700:Kinetics; D008928:Mitochondria / Q000201:enzymology; D009206:Myocardium / Q000201:enzymology; D011233:Precipitin Tests; D011768:Pyruvate Dehydrogenase Complex / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000378:metabolism*; D015325:Pyruvate Dehydrogenase Complex Deficiency Disease / Q000201:enzymology; D017670:Sodium Compounds / Q000494:pharmacology
D000911:Antibodies, Monoclonal; D018053:Arsenites; D011768:Pyruvate Dehydrogenase Complex; D017670:Sodium Compounds; C017947:sodium arsenite; D000255:Adenosine Triphosphate