Treatment of P. falciparum in vitro cultures with 0.5 mM limonene and metabolic labeling with [³H]GGPP and [³H]FPP. Parasites were treated for 24 h in the presence (+) or absence (−) of 0.5 mM limonene and labeled for 18 h with 6.25 μCi of [³H]GGPP (A) or [³H]FPP (B) per ml, lysed, and analyzed by SDS-PAGE (12.5% acrylamide) and fluorography. R, ring forms; T, trophozoites; S, schizonts. Lanes 1 in panels A and B show identically processed material from noninfected erythrocytes. Molecular mass (M_r) standards are indicated on the left. (C) Parasites were treated for 20 h with 0.5 mM limonene (+) or left untreated (−) and labeled with [³⁵S]methionine for 18 h, harvested, washed, lysed, and analyzed by SDS-PAGE (12.5% acrylamide). (D) Radiochromatogram of chloroform-methanol (9:1)-extractable P. falciparum proteins and products derived from [³H]FPP- or [³H]GGPP-labeled P. falciparum proteins resolved by SDS-PAGE that were resolved by RP-TLC. Lanes: 1, moieties cleaved from 21- to 24-kDa proteins labeled with [³H]GGPP; 2, moieties cleaved from 21- to 24-kDa proteins labeled with [³H]FPP; 3, control containing cleaved material from the 21- to 24-kDa region of noninfected, [³H]GGPP-labeled red blood cells. Standards run in parallel are indicated on the left. FOH, farnesol; GGOH, geranylgeraniol; ori, origin.

Immunoprecipitation of p21^ras from P. falciparum at the schizont stage. (A) Infected or noninfected red blood cells were labeled with [³H]FPP or [³H]GGPP and immunoprecipitated with anti-p20^h-rap/krev (lanes 1 and 3) or anti-p21^h-ras antibody (lanes 2 and 4). Lanes 3 and 4 show immunoprecipitation of noninfected red blood cells with the respective antibodies. (B and C) Limonene at 0.5 mM specifically inhibits protein p21^h-ras prenylation but not protein synthesis. (B) Identical numbers of purified lysed schizonts were labeled with [³H]FPP, treated (+) with limonene (0.5 mM) or left untreated (−), and then immunoprecipitated with protein A-Sepharose-bound monoclonal anti-Ras immunoglobulins and analyzed by SDS-PAGE and autoradiography. Lanes: 1, untreated parasites; 2, treated parasites. (C) Identical numbers of purified lysed schizonts labeled with [³⁵S]methionine and treated (+) with limonene (0.5 mM) or left untreated (−) were then immunoprecipitated with protein A-Sepharose-bound monoclonal anti-Ras immunoglobulins and analyzed by SDS-PAGE. Lanes: 1, untreated parasites; 2, treated parasites. The arrowhead indicates the molecular size range where the Ras-like protein is expected.

Monoterpene treatment partially arrests P. falciparum development at the ring stage. Parasite forms from cultures treated with 0.5 mM limonene and from untreated cultures were plotted against time of culture. The value for each form is given as a percentage of the sum of all forms at each time interval from three independent experiments. C, control cultures; L, limonene-treated cultures. The error bars indicate standard deviations. Significantly different values (P < 0.05) for treated versus untreated cultures are labeled with stars. Black stars indicate significant differences in treated or untreated ring-stage parasites, and white stars indicate significant differences in treated or untreated trophozoite-stage parasites. IRBC, infected red blood cells.