Microassay of decarboxylation reactions in cultured cells.
The currently described methods for determination of decarboxylation reaction rates in cultured cells require large quantities of cells and often involve cell manipulation prior to assay. We describe a simple microassay for the rapid measurement of various decarboxylation reaction rates in intact cultured cells. The assay is based on the traditional measurement of 14CO2 generated from 14C-labeled substrates. Key to the method is a novel modification of the standard petri dish. Pyruvate dehydrogenase, branched chain alpha-ketoacid dehydrogenase, alpha-ketoglutarate dehydrogenase, and ornithine decarboxylase activities were determined in adult cardiomyocyte cultures containing only 0.1-0.5 mg of protein per culture dish. Efficiency of 14CO2 collection ranged between 94 and 100%. Pharmacological enhancement or inhibition of pyruvate dehydrogenase activity was easily detected in the culture system. This new method simplifies the measurement of various decarboxylation reaction rates in cultured cells and allows rapid, reproducible measurements to be made on small numbers of cells without perturbation of the culture conditions or the cells themselves.
Bartos
D
D
Department of Surgery, Oregon Health Sciences University, Portland 97201.
Vlessis
A A
AA
Muller
P
P
Mela-Riker
L
L
Trunkey
D D
DD
D000818:Animals; D002245:Carbon Dioxide / Q000032:analysis* / Q000378:metabolism; D002250:Carbon Radioisotopes; D002478:Cells, Cultured; D003653:Decarboxylation; D007700:Kinetics; D008297:Male; D009206:Myocardium / Q000166:cytology* / Q000201:enzymology; D009955:Ornithine Decarboxylase / Q000378:metabolism*; D010088:Oxidoreductases / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D015203:Reproducibility of Results
D002250:Carbon Radioisotopes; D002245:Carbon Dioxide; D010088:Oxidoreductases; D009955:Ornithine Decarboxylase