Table 1.

Purification of the ceramide synthase

Fraction	Protein	Ceramide synthase activity	Yield	Enrichment
	mg/ml	nmol/mg of protein/min	Percentage	Fold
Digitonin-treated membranesa	5.6	6.1	100	1
Solubilized membranesb	4.6	6.0	80	0.98
Elution of anti-Flag M2 affinity beads	5.4 × 10−3c	20 678	68	3420
Ceramide synthase was purified from membranes as described in Materials and methods. Data are the average of five sets of experiments. Each time similar results were found.
aMembranes resuspended in the presence of 1% digitonin prior to incubation.
bSupernatant after centrifugation (100 000 g for 1 h) of the digitonin-treated membranes.
cProtein concentrations were estimated by densitometric comparison of silver-stained proteins of the fraction with stained calibration bands of bovine serum albumin of known concentrations in SDS–PAGE.