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Free keywords:
metabolism; enzyme activities; enzymatic cycling; glutaminolysis; MDCK
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.
Copyright 2010 Wiley Periodicals, Inc.
[accessed September 21st, 2010]
