杠柳新苷类化合物对东方粘虫和小地老虎幼虫中肠蛋白酶活性的影响
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摘要
杠柳新苷E、P和T是从杠柳根皮中分离到的结构相似的化合物，但杠柳新苷E无杀虫活性，杠柳新苷P具有强杀虫活性，而杠柳新苷T具有更强的杀虫活性。为了深入研究杠柳新苷类化合物的作用机理，本研究以丝氨酸蛋白酶抑制剂PMSF为抑制剂对照，比较研究了3种供试化合物分别对东方粘虫Mythimna separata和小地老虎Agrotisypsilon幼虫中肠类胰蛋白酶和类胰凝乳蛋白酶活性的影响；并进一步分离纯化粘虫中肠类胰蛋白酶，测定其相关性质，研究三种杠柳新苷类化合物对纯化后粘虫类胰蛋白酶活性的影响，进而探讨杠柳新苷类杀虫活性物质对粘虫的杀虫作用机理。主要结果如下：
1.杠柳新苷P和T对粘虫类胰蛋白酶，特别是对弱碱性类胰蛋白酶具强激活作用，药后2h激活率分别为167.78%和277.55%，激活时间分别长达8h和24h；两化合物在药后2h对小地老虎类胰蛋白酶有弱激活作用，药后其它时间均有不同程度的抑制作用，但抑制率较低；对粘虫和小地老虎类胰凝乳蛋白酶均无明显影响。
杠柳新苷E对粘虫中肠类胰蛋白酶仅表现出抑制作用，对小地老虎类胰蛋白酶无明显影响；此化合物对粘虫和小地老虎类胰凝乳蛋白酶均无显著影响。
2．从粘虫中肠分离到的类胰蛋白酶的分子量为25078Da，等电点为7.0。最适反应温度为45-50℃，且在低于45℃下热稳定性好。粘虫类胰蛋白酶的最大活力范围在pH10.0附近，pH值稳定性范围为8.5-10.5。以BApNA为底物，粘虫胰蛋白酶的Km值为0.18±0.0045mmol/L，Kcat值为86.25s~-1。
3.杠柳新苷P和T对纯化的粘虫类胰蛋白酶有弱激活作用。杠柳新苷T对纯化后的粘虫类胰蛋白酶活性的影响与杠柳新苷P相当，二者无显著差异。杠柳新苷E对纯化后的粘虫类胰蛋白酶没有明显影响。
这些结果表明，杠柳新苷P和T对粘虫类胰蛋白酶，特别是弱碱性类胰蛋白酶的强激活作用可能是造成粘虫中毒的作用机理之一。
Periplocoside E, P, T, which were isolated from the root bark of the insecticidal plantPeriploca sepium Bunge, are analogical compounds in structure. But, periplocoside E has noinsecticidal activity, periplocoside P has high insecticidal activity, while periplocoside T hasstronger insecticidal activity than periplocoside P. To explore the insecticidal mechanism ofperiplocosides compounds, we compared different effects on the activities of trypsin-likeprotease, chymotrypsin-like protease in the midgut of the6th instar larvae of Mythimnaseparata and Agrotis ypsilon among the three kinds of periplocosides compounds, take serineproteinase inhibitor PMSF as agent control. To explore the insecticidal mechanism ofinsecticidal periplocosides compounds, the trypsin-like protease in the midgut of the6th instarlarvae of M. separata were purification, the related properties of purified trypsin-like proteasewere assayed, and the effects on purified trypsin-like protease of M. separata of the threeperiplocosides compounds were studied. The main results were as follows:
1. Periplocoside P and T displayed strongly activated activity on trypsin-like protease,especially the weakly alkaline trypsin-like protease in the midgut of M. separata larvae. Theactivation ratio were167.78%and277.55%after2h of treatment, respectively, and theactivation time last up to8h and24h, respectively. Treatment by periplocoside P and T after2h, the trypsin-like protease activity in the midgut of A. ypsilon was weakly activated, whileother time after treatment, enzyme activity were inhibited, but the inhibition ratio were low.Periplocoside P and T showed no obvious effect on trypsin-like protease andchymotrypsin-like protease activity in the midgut of A. ypsilon.
Periplocoside E displayed inhibition effects on the trypsin-like protease activity in themidgut of M. separata larvae, while no obvious effect on that of A. ypsilon. Periplocoside Eshowed no obvious effect on chymotrypsin-like protease activity in the midgut of M. separataand A. ypsilon.
2. The molecular masses(MM) of purified trypsin from M. separata midgut was25,078Da. IP of purified trypsin showed to be7.0. The optimum temperature of purified trypsin werefound to be45-50℃. The purified trypsin was found to be heat stable over a wide range oftemperature(<45℃). The maximum activity for purified trypsin-like of M. separata wasrecorded at pH10.0. The pH-stability were shown at the pH rang8.5-10.5. The Km value forBApNA hydrolysis was0.18±0.0045mmol/L for purified trypsin of M. separata, and the Kcatvalue was86.25s~-1.
3. Periplocoside P and T displayed weakly activated activity on the purified trypsin-likefrom the midgut of M. separata larvae, which didn’t show concentration dependence. Therewas no significant activity difference on purified trypsin-like from the midgut of M. separatalarvae between periplocoside P and T. Periplocosides E showed no obvious effect on purifiedtrypsin-like, the dependence of concentration were not observed.
All of the results showed that the strong activation of trypsin-like in the midgut of M.separata may be one of the factors that periplocoside P and T cause toxicity to insects.
1.杠柳新苷P和T对粘虫类胰蛋白酶，特别是对弱碱性类胰蛋白酶具强激活作用，药后2h激活率分别为167.78%和277.55%，激活时间分别长达8h和24h；两化合物在药后2h对小地老虎类胰蛋白酶有弱激活作用，药后其它时间均有不同程度的抑制作用，但抑制率较低；对粘虫和小地老虎类胰凝乳蛋白酶均无明显影响。
杠柳新苷E对粘虫中肠类胰蛋白酶仅表现出抑制作用，对小地老虎类胰蛋白酶无明显影响；此化合物对粘虫和小地老虎类胰凝乳蛋白酶均无显著影响。
2．从粘虫中肠分离到的类胰蛋白酶的分子量为25078Da，等电点为7.0。最适反应温度为45-50℃，且在低于45℃下热稳定性好。粘虫类胰蛋白酶的最大活力范围在pH10.0附近，pH值稳定性范围为8.5-10.5。以BApNA为底物，粘虫胰蛋白酶的Km值为0.18±0.0045mmol/L，Kcat值为86.25s~-1。
3.杠柳新苷P和T对纯化的粘虫类胰蛋白酶有弱激活作用。杠柳新苷T对纯化后的粘虫类胰蛋白酶活性的影响与杠柳新苷P相当，二者无显著差异。杠柳新苷E对纯化后的粘虫类胰蛋白酶没有明显影响。
这些结果表明，杠柳新苷P和T对粘虫类胰蛋白酶，特别是弱碱性类胰蛋白酶的强激活作用可能是造成粘虫中毒的作用机理之一。
Periplocoside E, P, T, which were isolated from the root bark of the insecticidal plantPeriploca sepium Bunge, are analogical compounds in structure. But, periplocoside E has noinsecticidal activity, periplocoside P has high insecticidal activity, while periplocoside T hasstronger insecticidal activity than periplocoside P. To explore the insecticidal mechanism ofperiplocosides compounds, we compared different effects on the activities of trypsin-likeprotease, chymotrypsin-like protease in the midgut of the6th instar larvae of Mythimnaseparata and Agrotis ypsilon among the three kinds of periplocosides compounds, take serineproteinase inhibitor PMSF as agent control. To explore the insecticidal mechanism ofinsecticidal periplocosides compounds, the trypsin-like protease in the midgut of the6th instarlarvae of M. separata were purification, the related properties of purified trypsin-like proteasewere assayed, and the effects on purified trypsin-like protease of M. separata of the threeperiplocosides compounds were studied. The main results were as follows:
1. Periplocoside P and T displayed strongly activated activity on trypsin-like protease,especially the weakly alkaline trypsin-like protease in the midgut of M. separata larvae. Theactivation ratio were167.78%and277.55%after2h of treatment, respectively, and theactivation time last up to8h and24h, respectively. Treatment by periplocoside P and T after2h, the trypsin-like protease activity in the midgut of A. ypsilon was weakly activated, whileother time after treatment, enzyme activity were inhibited, but the inhibition ratio were low.Periplocoside P and T showed no obvious effect on trypsin-like protease andchymotrypsin-like protease activity in the midgut of A. ypsilon.
Periplocoside E displayed inhibition effects on the trypsin-like protease activity in themidgut of M. separata larvae, while no obvious effect on that of A. ypsilon. Periplocoside Eshowed no obvious effect on chymotrypsin-like protease activity in the midgut of M. separataand A. ypsilon.
2. The molecular masses(MM) of purified trypsin from M. separata midgut was25,078Da. IP of purified trypsin showed to be7.0. The optimum temperature of purified trypsin werefound to be45-50℃. The purified trypsin was found to be heat stable over a wide range oftemperature(<45℃). The maximum activity for purified trypsin-like of M. separata wasrecorded at pH10.0. The pH-stability were shown at the pH rang8.5-10.5. The Km value forBApNA hydrolysis was0.18±0.0045mmol/L for purified trypsin of M. separata, and the Kcatvalue was86.25s~-1.
3. Periplocoside P and T displayed weakly activated activity on the purified trypsin-likefrom the midgut of M. separata larvae, which didn’t show concentration dependence. Therewas no significant activity difference on purified trypsin-like from the midgut of M. separatalarvae between periplocoside P and T. Periplocosides E showed no obvious effect on purifiedtrypsin-like, the dependence of concentration were not observed.
All of the results showed that the strong activation of trypsin-like in the midgut of M.separata may be one of the factors that periplocoside P and T cause toxicity to insects.
引文
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