Figure 3
Biochemical properties of Nilaparvata lugens nCDase (NlnCDase). (A) Microsomes isolated from High Five cells transfected with pFast-HTB or pFast-HTB/NlnCDase were assayed for ceramidase activity. (B) The microsomes and supernatant of pFast-HTB/NlnCDase were subjected on SDS-PAGE gel and analyzed by western blotting. The relative Mr. was estimated according to protein standards. (C) The NlnCDase activity was assayed at different pH values. The pH was adjusted by adding the following buffer: Acetate (pH 2–6), Tris (pH 7–8), Glycine (pH 9–13). Ceramidase activity of the NlnCDase at each pH was calculated by subtracting ceramidase activity in pFast-HTB microsomes from that in pFast-HTB/NlnCDase microsomes. The NlnCDase activity at pH 6.0 was highest and sets as 100%, and ceramidase activity at other pH values was expressed as % of the maximal activity. (D) Optimum temperature of NlnCDase. The NlnCDase activity at each temperature was conducted at pH 6.0. The NlnCDase activity at 36 °C was highest and set as 100%, and ceramidase activity at other temperature was expressed as % of the maximal activity. (E) Effects of different cations on NlnCDase activity. 5 mM indicated cations were added in reaction buffer. The NlnCDase activity with Ca2+ added was highest and set as 100%, and ceramidase activity with different cations values was expressed as % of the maximal activity. (F) Substrate specificity of NlnCDase. The NlnCDase activity was highest with substrate of C12 ceramide and set as 100%, and ceramidase activity on other substrates was expressed as % of the maximum activity. All data represent the mean value ± SE of three independent experiments performed in duplicate, and the data with different letters are significantly different (two-samples t test, P < 0.05).
