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Measurement of Key Metabolic Enzyme Activities in Mammalian Cells Using Rapid and Sensitive Microplate-Based Assays

MPG-Autoren
http://pubman.mpdl.mpg.de/cone/persons/resource/persons86337

Janke,  R.
Bioprocess Engineering, Max Planck Institute for Dynamics of Complex Technical Systems, Max Planck Society;

http://pubman.mpdl.mpg.de/cone/persons/resource/persons86303

Genzel,  Y.
Bioprocess Engineering, Max Planck Institute for Dynamics of Complex Technical Systems, Max Planck Society;

http://pubman.mpdl.mpg.de/cone/persons/resource/persons86512

Wahl,  A.
Bioprocess Engineering, Max Planck Institute for Dynamics of Complex Technical Systems, Max Planck Society;

http://pubman.mpdl.mpg.de/cone/persons/resource/persons86448

Reichl,  U.
Otto-von-Guericke-Universität Magdeburg;
Bioprocess Engineering, Max Planck Institute for Dynamics of Complex Technical Systems, Max Planck Society;

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Zitation

Janke, R., Genzel, Y., Wahl, A., & Reichl, U. (2010). Measurement of Key Metabolic Enzyme Activities in Mammalian Cells Using Rapid and Sensitive Microplate-Based Assays. Biotechnology and Bioengineering, 107(3), 566-581. doi:10.1002/bit.22817.


Zitierlink: http://hdl.handle.net/11858/00-001M-0000-0013-90AD-F
Zusammenfassung
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. Copyright 2010 Wiley Periodicals, Inc. [accessed September 21st, 2010]