Figure 5.

Acyl chain specificity of CerS5^{(299–309→CerS2)}. A, homogenates were prepared from WT HEK cells overexpressing CerS5^{(299–309→CerS2)}, and activity was assayed using the indicated acyl-CoAs with increasing amounts of protein for 20 min. Results are means ± S.E. of two individual experiments performed in duplicate. B, homogenates (40 μg of protein) were prepared from WT HEK cells overexpressing CerS2 (triangles), CerS5 (circles), or CerS5^{(299–309→CerS2)} (squares), and activity was assayed using C22-CoA for 30 min. Results are from a typical experiment in which CerS activity was assayed in duplicate and repeated four times. Levels of expression of all CerS constructs were similar as determined by Western blotting. C, fatty acid composition of ceramide was determined in WT HEK cells overexpressing pcDNA (black) or CerS5^{(299–309→CerS2)} (gray). n = 6. *, p < 0.05; **, p < 0.01.