Fig. 4.

Acyl-CoA donor specificity and temperature preference of Y-strain epimastigotes of T. cruzi CerS in vitro. (A) [³H]SPH was incubated in 100 μL of 100 mM Tris-HCl pH 7.5 containing 1 mM MgCl₂, 1 mM DTT, 4 mM NaF and 0.2% (w/v) CHAPS at 28 °C (lanes 1–11) in the absence (−) or presence of epimastigote microsomes (50 μg·mL⁻¹) and in the absence (−) or presence (+) of various acyl-CoAs of different lengths (100 μM). (B) Lanes 12–17 are the same as lanes 1–11, but only palmitoyl-CoA was used as the acyl-CoA substrate donor; 200 μg·mL⁻¹ of microsomes were present, and the temperature was varied between 14 °C, 28 °C and 37 °C. After 4 h incubation, lipids were extracted and analyzed by TLC as in Figure 2. The relative positions of palmitoyl-sphingosine (C16-Cer) and lignoceroyl-sphingosine (C24-Cer) standards are indicated on the left of Panel A.