Figure 4. Stearoyl-CoA desaturase activity in a P. falciparum cell-free assay.

(A) In vitro conversion of stearoyl-CoA to oleoyl-CoA in P. falciparum 3D7 schizont lysates, measured as a function of protein concentration. The reaction mix contained 0 to 2 mg of total lysate proteins, [¹⁴C]-stearoyl-CoA, and 2 mM NADPH, and was incubated for 30 min at 37°C. Following extraction of FAMEs, equal volumes of each sample (30%) were loaded on TLC plates and resolved using petroleum ether/diethyl ether (17/3, v/v). Radiolabeled FAMEs were revealed after overnight exposure to a Kodak Biomax MR film. Spots corresponding to oleic acid were scraped from the TLC plate and counted. Results are expressed as picomoles of oleic acid produced. (B) In vitro conversion of stearoyl-CoA as a function of reaction time. The reaction mix containing 500 µg total proteins, [¹⁴C]-stearoyl-CoA, and 2 mM NADPH, and was incubated at 37°C for various durations. Following extraction of FAMEs, equal volumes of each sample (25%) were loaded on TLC plates, resolved and revealed as described above. Results are expressed as picomoles of oleic acid produced. (C) In vitro conversion of stearoyl-CoA as a function of the substrate concentration. Increasing concentrations of [¹⁴C]-stearoyl-CoA, ranging from 2 to 200 µM, were mixed together with 500 µg total protein and 2 mM NADPH, and the reactions incubated for 5 min at 37°C. Following extraction of FAMEs, equal volumes of each sample (20%) were loaded on a TLC plate, resolved and revealed as described above. Oleic acid methyl esters were scraped from the TLC plate and counted to allow direct quantification of the enzymatic reaction. The specific activity is expressed in picomoles of oleoyl-CoA formed/hr/mg of input protein. The inset corresponds to the Lineweaver-Burke plot of the results. Results are expressed as a mean of 2 independent experiments. (D) NADPH dependency of the Stearoyl-CoA desaturase activity. Schizont membranes were incubated with [¹⁴C]-stearoyl-CoA either in the absence (−) or presence of 2 mM NADH or NADPH. Samples were processed as described in (A) and FAMEs were analyzed by TLC/autroradiography. Each condition was performed in duplicate.