FIGURE 2.

Ceramide is produced from sphingosine and palmitoyl-CoA by purified rat liver mitochondria at isotonic conditions. Panel A, ceramide formation as a function of protein concentration is shown. Mitochondria at the indicated amounts were incubated as described under “Experimental Procedures” in the presence of 50 μm palmitoyl-CoA and 15 μm sphingosine for 15 min before termination of the reaction by addition of ethyl acetate/isopropyl alcohol extraction mixture. Where indicated, Triton X-100 (final concentration 0.5%) was added simultaneously with mitochondria. Panel B, absorption of mitochondrial suspension as a function of protein concentration in the absence or in the presence of 15 μm sphingosine is shown. Absorption was measured 15 min after sphingosine addition. Panel C, time course of ceramide formation is shown. Mitochondria (1 mg/ml) were incubated as described under “Experimental Procedures” in the presence of 50 μm palmitoyl-CoA and 15 μm sphingosine for the indicated times before termination of the reaction by the addition of ethyl acetate/isopropyl alcohol extraction mixture. Results represent the average ±S.D. (n = 3).