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Proteins including 5-LO were precipitated with 50% saturated
ammonium sulfate during stirring on ice for 60 min. The precipitate
was collected by centrifugation at 16000g for 25 min and the pellet
was resuspended in 20 mL of PBS containing 1 mM EDTA and 1
mM PMSF. After centrifugation at 100000g for 70 min at 4 °C,
the pellet was removed and the supernatant (S100) was used for
5-LO activity assays. ATP (1 mM) and the test compounds or
vehicle (DMSO, <0.3% vol/vol) were added to the S100, and after
5-10 min at 4 °C, samples were prewarmed for 30 s at 37 °C. To
start 5-LO product formation, 2 mM CaCl2 and 20 µM AA at the
indicated concentrations were added. The reaction was stopped after
10 min at 37 °C by addition of 1 mL of ice-cold methanol, and the
formed metabolites were analyzed by HPLC as described for intact
cells.
Statistics. All values are reported as mean ( SE. Statistical
comparisons were determined using the Student’s t-test for cor-
related samples (inhibitor versus control), and significance was
accepted at p < 0.05.
Acknowledgment. We thank Dr. Bernd Sorg for helpful
discussion and for critically reading of the manuscript. This
research was supported by the EU (Leuchron, QLRT-2000-
01521) and the Deutsche Forschungsgemeinschaft (GRK 137/
3).
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Supporting Information Available: Experimental details. This
material is available free of charge via the Internet at http://
pubs.acs.org.
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