FDPS activity assay

Briefly, FPPs was assayed in 150 µl containing 25 mM Hepes, pH = 7, 2 mM MgCl2, 1 mM dithiothreitol, 5 mM KF, 1% n-octyl-ß-glycopyranoside, 3.3 µM [4-14 C] IPP (18 Ci/mmol), 3 µM unlabeled IPP and 20 µM geranyl diphosphate. Reactions were started by adding 40 µl of peroxisomal fraction containing 100 µg of total protein and incubated for 45 min at 37 °C. Reactions were stopped by the addition of 150 µl 2.5 N HCl in 80% ethanol containing 100 µg/ml farnesol as a carrier. The samples were hydrolyzed for 30 min at 37 °C to convert the FPP to farnesol and neutralized by the addition of 150 µl of 10% NaOH. The reaction product (farnesol) was extracted into 1 ml of n-hexane and an aliquot (200 µl) of the organic phase was used for radioactivity counting. One unit of enzyme activity is defined as the amount of enzyme required to synthesize one pmol of FPP per min. Parallel samples were assayed to evaluate the total and the nonspecific radioactivity. In all experiments, enzyme assays were carried out in duplicate. The coefficient percentages of intra- and interassay variation were 3 and 4%, respectively.