Measurement of key metabolic enzyme activities in mammalian cells using rapid and sensitive microplate‐based assays

Abstract

Sensitive microplate-based assays to determine low levels of key enzyme activities in mammalian cells are presented. The enzyme platform consists of four cycling assays to measure the activity of 28 enzymes involved in central carbon and glutamine metabolism. The sensitivity limit of all cycling assays was between 0.025 and 0.4 nmol product. For the detection of glutaminase activity, a new glutamate cycle system involving the enzymes glutamate dehydrogenase and aspartate transaminase was established. The relative standard deviation of the method was found to be 1.7% with a limit of detection of 8.2 pmol and a limit of quantitation of 24.8 pmol. Hence, cell extracts could be highly diluted to reduce interferences caused by other components in the extract, which in addition minimized underestimates or overestimates of actual enzyme activities. Since substrate concentrations could be maintained at a nearly constant level throughout the assay product accumulation during the reaction was low, which minimized product inhibition. As an example, the enzyme platform was used to investigate maximum enzyme activities of stationary-phase MDCK cells grown in serum-containing GMEM medium as typically used in influenza vaccine production.

Inhibition of Mitochondrial Pyruvate Transport by Zaprinast Causes Massive Accumulation of Aspartate at the Expense of Glutamate in the Retina

“…Pyruvate Dehydrogenase Activity (PDH) Activity Assay-PDH activity was measured using an NADH cycling assay in the presence of diaphorase to reduce the generated NADH and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was as an electron acceptor (27). Purified PDH protein from pig heart was incubated with 1 mM MgCl 2 , 5 mM CaCl 2 , 5 mM l-carnitine, 0.2 mM thiamine pyrophosphate, 0.1 mM CoA, 2.5 mM NAD ϩ , 1 mM MTT, and 1 unit/ml diaphorase in 50 mM HEPES/KOH buffer, pH 7.5, at 30°C.…”

PPARα and fatty acid oxidation mediate glucocorticoid resistance in chronic lymphocytic leukemia

“…The assay was conducted according to the protocol of Janke et al 12,13 ATP produced by PK from ADP and phosphoenolpyruvate was fed into the glycerol-3-phosphate cycling assay by glycerol kinase. Optimal conditions for measuring glycerol-3-phosphate levels to indicate maximal PK activity included a cell dilution factor (CDF) of 150, pH 7.0, and 60-minute incubation time.…”

Microplate based assay for thrombin detection using an RNA aptamer as affinity ligand and cleavage of a chromogenic or a fluorogenic peptide substrate

“…The microplate-based assays offer benefits in terms of fast sample handling, analysis of multiple samples, small volume, continuous monitoring and convenience, which are beneficial to the practical application [21,22]. A few groups reported the assays for thrombin by using microplates as solid support and DNA aptamers as affinity ligands in different formats [23][24][25][26][27][28].…”