Department of Plant Protection, College of Horticultural Sciences and Plant Protection, University College of Agriculture and Natural Resources, University of Tehran, Karaj, Iran.
J Insect Sci. 2009;9:1-11. doi: 10.1673/031.009.7001.
The Sunn pest, Eurygaster integriceps Puton (Heteroptera: Scutelleridae), is one of the most important pests of wheat and causes considerable damage to this valuable crop annually. Digestive proteinase activity of adult insects was investigated using general and specific substrates and inhibitors. Proteolytic activity was low when the common conventional substrates, azoalbumin, azocasein and hemoglobin were used to assay salivary glands and midguts. Using the fluorescent casein substrate (BODIPY FL casein), total proteolytic activity was measured at different pH. Maximum proteolytic activity was detected at pH 7 (100%) and 8(65%) which suggested the presence of serine proteinases in the salivary glands. There was no detectable proteolytic activity in midgut extracts. The inhibitors; PMSF (inhibitor of serine proteinases) and TPCK (a specific chymotrypsin inhibitor) showed greater than 50% inhibitory effect on total proteolytic activity, however, TLCK (specific trypsin inhibitor) and E-64(specific cysteine proteinase inhibitor) did not inhibit total proteolytic activity. Using fluorescent specific substrates for serine and cysteine proteinases (Z-Arg-AMC, Z-Arg-Arg-AMC, Z-Arg-Phe-AMC and Suc-Ala-Ala-Pro-Phe-AMZ) revealed the presence of tryptic and chymotryptic activity in the salivary gland extract. Zymogram analysis under non-reducing SDS-PAGE conditions and using the substrate APNE showed at least 8 tryptic and chymotryptic activity bands in salivary gland extracts. A single high molecular weight band with tryptic activity (165 kDa) was detected using the substrate BApNA in a zymogram analysis using native-PAGE. Kinetic studies showed a k(m) value of 0.6 mM for this enzyme against the substrate BApNA .The inhibitor TLCK decreased activity of the trypsin-like enzyme up to 73% and almost completely eliminated the only band related to this proteinase in the zymogram. Soybean Kunitz type trypsin inhibitor showed no effect on proteolytic activity of the trypsin-like serine proteinase. In general, the results revealed the presence of chymotrypsin- and trypsin-like serine proteinases in the salivary gland of E. integriceps, and it seems that the major total proteolytic activity is due to chymotrypsin proteinases.
麦圆叶爪螨，Eurygaster integriceps Puton（半翅目：叶甲科），是小麦的重要害虫之一，每年都会对这种有价值的作物造成相当大的损害。使用通用和特定的底物和抑制剂研究了成虫的消化蛋白酶活性。当使用常见的常规底物，偶氮白蛋白、偶氮酪蛋白和血红蛋白来测定唾液腺和中肠时，蛋白酶活性很低。使用荧光酪蛋白底物（BODIPY FL 酪蛋白），在不同 pH 值下测量总蛋白水解酶活性。在 pH 7（100%）和 8（65%）时检测到最大的蛋白水解酶活性，这表明唾液腺中存在丝氨酸蛋白酶。中肠提取物中没有检测到可检测的蛋白水解酶活性。抑制剂；PMSF（丝氨酸蛋白酶抑制剂）和 TPCK（一种特异性糜蛋白酶抑制剂）对总蛋白水解酶活性的抑制作用大于 50%，然而，TLCK（特异性胰蛋白酶抑制剂）和 E-64（特异性半胱氨酸蛋白酶抑制剂）没有抑制总蛋白水解酶活性。使用荧光特异性丝氨酸和半胱氨酸蛋白酶底物（Z-Arg-AMC、Z-Arg-Arg-AMC、Z-Arg-Phe-AMC 和 Suc-Ala-Ala-Pro-Phe-AMZ）显示唾液腺提取物中存在胰蛋白酶和糜蛋白酶活性。在非还原 SDS-PAGE 条件下进行的酶谱分析和使用底物 APNE 显示，在唾液腺提取物中至少有 8 种胰蛋白酶和糜蛋白酶活性带。在使用天然-PAGE 的底物 BApNA 的酶谱分析中，检测到一种具有胰蛋白酶活性的单一高分子量带（165 kDa）。动力学研究表明，该酶对底物 BApNA 的 k(m) 值为 0.6 mM。抑制剂 TLCK 使胰蛋白酶样酶的活性降低了 73%，并几乎完全消除了酶谱中与该蛋白酶相关的唯一带。大豆 Kunitz 型胰蛋白酶抑制剂对胰蛋白酶样丝氨酸蛋白酶的蛋白水解活性没有影响。总的来说，结果表明 E. integriceps 的唾液腺中存在胰蛋白酶和糜蛋白酶样丝氨酸蛋白酶，而且主要的总蛋白水解酶活性似乎是由糜蛋白酶蛋白水解酶引起的。
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