FIG. 2.

TLC analysis of products synthesized by M. tuberculosis (A) or M. smegmatis (B) cytosol in the presence of [¹⁴C]IPP and GPP. Prenyl diphosphate synthase activity was assayed in mixtures containing 50 mM MOPS (pH 7.9), 10 mM sodium orthovanadate, 5 mM MgCl₂, 2.5 mM dithiothreitol, 0.3% Triton X-100, 100 μM allylic diphosphate, 30 μM [¹⁴C]IPP, and 300 μg of protein in a final volume of 200 μl. Reaction mixtures were incubated at 37°C for 60 min (A) or 10 min (B). The reactions were stopped by the addition of 1 ml of water saturated with NaCl, and the product was extracted with n-butanol saturated with water. The resulting prenyl diphosphates were dephosphorylated with potato acid phosphatase, and equivalent amounts of radioactivity derived from dephosphorylated products were analyzed on LKC₁₈F TLC plates developed in methanol-acetone (8:2, vol/vol). Radioactive spots were located with a System 200 Imaging Scanner (Bioscan Inc.), and standard polyprenols were located with an anisaldehyde spray reagent (9).