Figure 2. Biochemical characterization of the human neutral CDase expressed in yeast.
(A) The effect of different amounts of yeast protein on CDase activity at different times. (B) Different amounts of human neutral CDase were assayed by incubation at 37 °C for 1 h as described in the Experimental section. For (C) and (D), 60 μg of yeast microsomal protein was used for CDase activity assay and the reaction time was 1 h. (C) Michaelis–Menten representations for CDase activity toward increasing concentrations of NBD-C12-ceramide. (D) pH optimum determination. The buffers used are described in the Experimental section. The results shown are representative of triplicates from at least two independent experiments. Abbreviation: F. Units, arbitrary fluorescence units.