A coupled fluorometric rate assay for pyruvate dehydrogenase in cultured human fibroblasts.
A method for measuring the activity of the pyruvate dehydrogenase complex (PDC) by coupling acetyl-CoA production to acetylation of a fluorescent dye is described. Acetylation of cresyl violet acetate by pigeon liver acetyltransferase results in a shift of its fluorescence spectrum from lambda ex max = 575, lambda em max = 620 nm to lambda ex max = 475, lambda em max = 575 nm. The rate of appearance of acetylated dye was followed fluorometrically and was proportional to PDC activity in extracts of cultured human fibroblasts. The assay showed appropriate substrate and cofactor dependence and had a working range between 0.04 and 70 munits. It is 10 times more sensitive than the spectrophotometric assay on which it is based (working range 0.4-31 munits) and is equally convenient. Unactivated PDC activity in fibroblast extracts was 0.75 (0.60-0.92) munits/mg protein (mean and range for six cell lines).
D000105:Acetyl Coenzyme A; D000818:Animals; D048588:Benzoxazines; D002478:Cells, Cultured; D055598:Chemical Phenomena; D002621:Chemistry; D002855:Chromatography, Thin Layer; D005347:Fibroblasts / Q000201:enzymology*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007223:Infant; D008297:Male; D009206:Myocardium / Q000201:enzymology; D010078:Oxazines; D011768:Pyruvate Dehydrogenase Complex / Q000302:isolation & purification*; D013050:Spectrometry, Fluorescence; D013552:Swine
D048588:Benzoxazines; D010078:Oxazines; D011768:Pyruvate Dehydrogenase Complex; C028911:cresyl violet; D000105:Acetyl Coenzyme A