Figure 4. Effects of different protease inhibitors and chemical modifiers on CDase activity.

(A) The CDase assay was carried out in the presence of different protease inhibitors at the indicated concentrations for 1 h at 37 °C. (B–E) Chemical-modification reactions were carried out as described in the Experimental section. Control reactions contained no modifiers, but did contain enzyme. (B–C) Purified rat brain CDase enzyme was used for the assays: (B) effect of increasing concentration of DFP upon purified CDase activity; (C) effect of DEPC and NEM upon purified CDase activity. (D–E) Effect of pre-incubation of ceramide on the DFP inhibition of neutral CDase. The human neutral CDase was expressed in yeast and prepared as described in the Experimental section: (D) yeast lysates were preincubated with or without 100 μM DFP in 100 mM phosphate buffer, pH 7.5, at 37 °C for 15 min. (E) Yeast lysates were first preincubated with (500 μM) D-erythro-C₁₂-NBD-ceramide for 30 min on ice, and then incubated with or without 100 μM DFP for another 15 min at 37 °C. The values are expressed as means±S.D. (n=3). The results are representative of triplicates from two independent experiments.