1 Department of Biology, Faculty of sciences, University of Guilan, Rasht, Iran.
J Insect Sci. 2013;13:81. doi: 10.1673/031.013.8101.
Proteolytic activities in digestive system extracts from the larval midgut of the lesser mulberry pyralid, Glyphodes pyloalis Walker (Lepidoptera: Pyralidae), were analyzed using different specific peptide substrates and proteinase inhibitors. High proteolytic activities were found at pH 10.0 and a temperature of 50° C using azocasein as substrate. The trypsin was active in the pH range of 9.5- 12.0, with its maximum activity at pH 11.5. Ethylene diamine tetraacetic acid had the most inhibitory effect, and 44% inhibition was detected in the presence of this inhibitor. Phenyl methane sulfonyl floride and N-tosyl-L-phe chloromethyl ketone also showed considerable inhibition of larval azocaseinolytic activity, with 40.2 and 35.1% inhibition respectively. These data suggest that the midgut of larvae contains mainly metalloproteases and serine proteases, mainly chymotrypsin. The effect of several metal ions on the activity of proteases showed that NaCl, CaCl2, CoCl2 (5 and 10 mM), and MnCl2 (5mM) reduced the protease activity. The kinetic parameters of trypsin-like proteases using N-benzoyl-L-arg-p-nitroanilide as substrate indicated that the Km and Vmax values of trypsin in the alimentary canal were 50.5 ± 2.0 µM and 116.06 ± 1.96 nmol min(-1) mg(-1) protein, respectively. Inhibition assays showed only small amounts of cysteine proteases were present in the G. pyloalis digestive system. The midgut digestive protease system of G. pyloalis is as diverse as that of any of the other polyphagous lepidopteran insect species, and the midgut of larvae contains mainly metalloproteases. Moreover, serine proteases and chymotrypsin also play main roles in protein digestion. Characterization of the proteolytic properties of the digestive enzymes of G. pyloalis offers an opportunity for developing appropriate and effective pest management strategies via metalloproteases and chymotrypsin inhibitors.
利用不同的特异性肽底物和蛋白酶抑制剂分析了幼虫中肠消化液提取物中小桑野蚕 Glyphodes pyloalis Walker（鳞翅目：螟蛾科）的蛋白水解活性。使用偶氮酪蛋白作为底物时，在 pH 值为 10.0 和温度为 50°C 时发现具有高蛋白水解活性。胰蛋白酶在 pH 值 9.5-12.0 的范围内具有活性，在 pH 值 11.5 时具有最大活性。乙二胺四乙酸的抑制作用最大，在存在该抑制剂的情况下检测到 44%的抑制作用。苯甲脒磺酰氟和 N-甲苯磺酰-L-苯丙氨酰氯甲基酮也对幼虫偶氮酪蛋白水解活性表现出相当大的抑制作用，抑制率分别为 40.2%和 35.1%。这些数据表明幼虫中肠主要含有金属蛋白酶和丝氨酸蛋白酶，主要是糜蛋白酶。几种金属离子对蛋白酶活性的影响表明，NaCl、CaCl2、CoCl2（5 和 10mM）和 MnCl2（5mM）降低了蛋白酶活性。使用 N-苯甲酰-L-精氨酰对硝基苯胺作为底物的胰蛋白酶样蛋白酶的动力学参数表明，肠内胰蛋白酶的 Km 和 Vmax 值分别为 50.5±2.0µM 和 116.06±1.96 nmol min-1 mg-1 蛋白。抑制试验表明，G. pyloalis 消化系统中仅存在少量半胱氨酸蛋白酶。G. pyloalis 的中肠消化蛋白酶系统与任何其他多食性鳞翅目昆虫物种一样多样化，幼虫中肠主要含有金属蛋白酶。此外，丝氨酸蛋白酶和糜蛋白酶也在蛋白质消化中起主要作用。G. pyloalis 消化酶的蛋白水解特性的表征为通过金属蛋白酶和糜蛋白酶抑制剂开发适当和有效的害虫管理策略提供了机会。
