棉铃虫幼虫中肠微粒体P450差光谱与O-脱甲基酶的酶学特征及其可诱导性

O-DEMETHYLASE ACTIVITY OF COTTON BOLLWORM (HELICOVERPA ARMIGERA) LARVAE:OPTIMAL ASSAY CONDITIONS AND INDUCIBILITY BY PHENOBARBITAL

棉铃虫Helicoverpaarmigera(H櫣ｂｎｅｒ)幼虫中肠微粒体制备液的CO差光谱在450nm有吸收峰,P450含量为(687±11)pmol/mg。中肠750g离心的上清液的O脱甲基酶活性在酶量相当5个中肠、反应时间30min内与酶量和反应时间呈线性关系;最适pH值在78,最适温度为20～25℃。酶系对底物对硝基苯甲醚的O脱甲基活性的Km=123mmol/L,Vmax=254nmol对硝基酚/(mg·min)。NADPH为酶活的重要因子,离体测定时,O脱甲基酶在无外来NADPH的活性仅为加025mmol/LNADPH的16%。在反应体系中加入15%BSA明显促进产物的生成。P450的专一性抑制剂PBO浓度达到1mmol/L时,可抑制酶90%的活性。棉铃虫取食含025%苯巴比妥钠的食物72h后,O脱甲基酶活性是对照组的173倍。

CO difference spectrum of midgut microsome preparation with P450 content of (687±11)pmol/mgPr. showed absorbance peak at 450nm.O demethylase of p nitroanisile( p NA) was maximally active at pH 7 8 and temperature 20～25℃. Activity was linear with respect to time for up to 30min, and with respect to enzyme level for up to 5 tissue equivalents/ incubation with 750g supernatant as enzyme source. K m and V max of O demethylation of p NA estimated were 1 23mmol/L and 2 54nmol p nitrophenol formed/mgPr/min. respectively. NADPH was one of the requirements for the O demethylation of p NA, the activity without external NADPH is 16% as that of with 0 25mmol/L NADPH.1 5% BSA added in the incubation mixture enhanced (1 2 folds) the production of p nitrophenol. PBO (1 mmol/L) strikingly (90%) inhibited O demethylation.1 73 folds induction of O demethylase activity was observed by dietary 0 25% PB.