Abstract
A sensitive, nonradioactive analytical method has been developed to simultaneously determine the concentrations of farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP) in cultured cells. Following extraction, enzyme assays involving recombinant farnesyl protein transferase or geranylgeranyl protein transferase I are performed to conjugate FPP or GGPP to dansylated peptides. The reaction products are then separated and quantified by high-performance liquid chromatography coupled to a fluorescence detector at the excitation wavelength 335 nm and the emission wavelength 528 nm. The retention times for farnesyl–peptide and geranylgeranyl–peptide are 8.4 and 16.9 min, respectively. The lower limit of detection is 5 pg of FPP or GGPP (∼0.01 pmol). A linear response has been established over a range of 5–1000 pg (∼0.01–2 pmol) with good reproducibility. The method has been used to determine the levels of FPP (0.125 ± 0.010 pmol/10 6 cells) and GGPP (0.145 ± 0.008 pmol/10 6 cells) in NIH3T3 cells. Furthermore, changes in FPP and GGPP levels following treatment of cells with isoprenoid biosynthetic pathway inhibitors were measured. This method is suitable for the determination of the concentrations of FPP and GGPP in any cell type or tissue.
Published Version
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