虫酰肼对甜菜夜蛾酚氧化酶的抑制作用

【目的】研究证实虫酰肼是否对甜菜夜蛾(Spodoptera exigua)酚氧化酶活性具有抑制作用。【方法】采用生物化学方法,分别测定虫酰肼对甜菜夜蛾5龄幼虫离体酚氧化酶的作用特点和用虫酰肼浸泡饲料饲喂5龄幼虫后,虫酰肼对幼虫头部、血淋巴及表皮组织酚氧化酶活性的抑制作用。【结果】随着虫酰肼浓度增大,酚氧化酶活力呈逐渐下降趋势,虫酰肼对酚氧化酶的抑制中浓度(IC50)为5.43μmol·L-1。虫酰肼对酚氧化酶的抑制作用表现为混合型抑制,游离酶抑制常数(K i)和酶-底物络合物抑制常数(K is)分别为11.223μmol·L-1和34.036μmol·L-1。通过研究金属铜离子和酶液对虫酰肼吸收峰的影响,推测虫酰肼对甜菜夜蛾酚氧化酶的抑制作用不是与该酶活性中心的铜离子结合,而可能是与酚氧化酶蛋白结合发生作用有关。用28.41μmol·L-1和85.23μmol·L-1亚致死剂量浓度的虫酰肼分别处理甜菜夜蛾5龄幼虫24、48和72h后,测定其头部、血淋巴和表皮酚氧化酶的活性。结果表明,随着用虫酰肼处理时间延长,两个剂量的虫酰肼对幼虫头部、血淋巴和表皮酚氧化酶的抑制作用基本呈逐渐增大趋势;处理后相同时间,高剂量的虫酰肼对酚氧化酶活性的抑制率基本上高于低剂量的抑制率;虫酰肼对头部酚氧化酶的抑制率最高,血淋巴次之,表皮最低。【结论】虫酰肼对甜菜夜蛾酚氧化酶具有明显的抑制作用,幼虫新表皮形成过程受到阻碍可能与虫酰肼抑制酚氧化酶的活性有关。

Inhibitory Effects of Tebufenozide on Phenoloxidase from 5th Instar Larvae of Spodoptera exigua

[Objective] In this paper, the inhibitory effect of tebufenozide on phenoloxidase (PO) in 5th instar larvae of the common rmyworm, Spodoptera exigua, was determined. [Method] Fifth instar larvae of S. exigua were supplied with tebufenozide-treated artifical diet to eat for different times and the inhibitory effect of tebufenozide on PO extracted from tebufenozide-treated and non-treated 5th instar larvae were determined by biochemical methods. [Result] The results indicated that tebufenozide could inhibit PO activity obviously, and the inhibition concentration of tebufenozide mixed with PO directly leading to 50% activity loss (IC50) was 5.43 μmol·L-1. The inhibitory mechanism of tebufenozide on PO was a mixed-competitive inhibition for oxidation of -dihydroxybenzene, the dissociative enzraye inhibition constant (Ki) and E-S' inhibition constant (Kis) were 11.223 μmol·L-1 and 34.036μmol·L-1, respectively. This inhibition may carry out through tebufenozide directly combining with enzyme protein while may be not chelating with Cu2+ in the active center of PO. The PO activity in head, hemolymph and epidermis of 5th instar larvae was also significantly inhibited after the larvae were treated with two sublethal doses (28.41μmol·L-1 and 85.23 μmol·L-1) of tebufenozide for continuous 24, 48 and 72 h, respectively. The inhibition rates increased with tebufenozide-treated time increasing, and inhibition rates of 85.23 μmol·L-1 were almost higher than those of 28.41 μmol·L-1 in the same tissues at the same length of tebufenozide-treated times. The orders of inhibition rates of PO by tebufenozide in different tissues at the same length of times were head＞ hemolymph＞ epidermis. [ Conclusion ] Tebufenozide can significantly inhibited PO ctivity, and it may be infer that the interference of new cuticle developmemt of larvae by tebufenozide is closely related to the inhibition of PO activity by tebufenozide.