Microassay of decarboxylation reactions in cultured cells

Dagmar Bartos, Angelo A. Vlessis, Patrick Muller, Leena Mela-Riker, Donald D. Trunkey

Research output: Contribution to journalArticle

5 Scopus citations

Abstract

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 α-ketoacid dehydrogenase, α-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.

Original languageEnglish (US)
Pages (from-to)241-244
Number of pages4
JournalAnalytical Biochemistry
Volume213
Issue number2
DOIs
StatePublished - Sep 1993

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ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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