5.26 FTase and GGTase I assays

FTase and GGTase I activity was determined using the method of Temple et al.,³⁹ with some modifications. For FTase, reaction mixtures were initiated by the addition of 18 nM recombinant FTase (Jena Biosciences) to reaction buffer (50 mM HEPPSO, 5 mM TCEP-HCl, 5 mM MgCl₂) containing 10 μM FDP (Sigma) and 5 μM dansyl-GCVLS (Biosynthesis, Inc.). For GGTase I, 24 nM of recombinant GGTase I (Jena Biosciences) was added to reaction buffer (50 mM HEPPSO, 5 mM TCEP-HCl,) containing 10 μM GGDP (Sigma) and 5 μM dansyl-GCVLS (Biosynthesis, Inc.). Reactions were carried out at 30 °C in the presence or absence of test compounds for 1 hour (FTase) or 2 hours (GGTase I). Prenylation of the peptide results in an increase in fluorescence intensity of the dansyl group (λ_ex = 340 nm, λ_em = 520 nm). Changes in fluorescence over time were detected using a Molecular Devices Spectramax Gemini EM fluorescence microplate reader.