Elpidina E N, Vinokurov K S, Gromenko V A, Rudenskaya Y A, Dunaevsky Y E, Zhuzhikov D P
A.N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow, Russia.
Arch Insect Biochem Physiol. 2001 Dec;48(4):206-16. doi: 10.1002/arch.10000.
Compartmentalization of proteinases, amylases, and pH in the midgut of Nauphoeta cinerea Oliv. (Blattoptera:Blaberidae) was studied in order to understand the organization of protein and starch digestion. Total proteolytic activity measured with azocasein was maximal at pH 11.5 both in anterior (AM) and posterior (PM) halves of the midgut, but the bulk of activity (67%) was found in PM. Total AM and PM preparations were fractionated on a Sephadex G-50 column and further analysed by means of activity electrophoresis and specific inhibitors and activators. The major activity in PM was classified as an unusual SH-dependent proteinase with M(r) 24,000 and pH optimum with synthetic substrate BApNA at 10.0. The enzyme was 43-fold activated in the presence of 1 mM DTT, insensitive to synthetic inhibitors of serine (PMSF, TLCK, TPCK) and cysteine (IAA, E-64) proteinases, strongly inhibited by STI, and displayed four active bands on zymograms. In PM, activities of trypsin-like, chymotrypsin-like, subtilisin-like, and cysteine proteinases were observed. Aspartic and metalloproteinases were not detected. In AM, activity of unusual SH-dependent proteinase also dominated and activity of chymotrypsin-like proteinase was observed, but their levels were much lower than in PM. Distribution of amylase activity, exhibiting an optimum at pH 6.0, was quite the opposite. The major part of it (67%) was located in AM. Treatment of amylase preparation with proteinases from AM and PM reduced amylase activity twofold. pH of the midgut contents was 6.0-7.2 in AM, 6.4-7.6 in the first and 8.8-9.3 in the second halves of PM. Thus, pH in AM is in good agreement with the optimal pH of amylase, located in this compartment, but the activity of proteinases, including the ability to degrade amylase, in such an environment is low. Active proteolysis takes place in the second half of PM, where pH of the gut is close to the optimal pH of proteinases.
为了解蛋白质和淀粉消化的组织情况，对灰翅夜蛾（Nauphoeta cinerea Oliv.，蜚蠊目：硕蠊科）中肠内蛋白酶、淀粉酶和pH值的区室化进行了研究。用偶氮酪蛋白测定的总蛋白水解活性在中肠前半部分（AM）和后半部分（PM）的pH 11.5时均达到最大值，但大部分活性（67%）存在于PM中。将AM和PM的总制剂在Sephadex G - 50柱上进行分级分离，并通过活性电泳以及特异性抑制剂和激活剂进行进一步分析。PM中的主要活性被归类为一种不寻常的依赖SH的蛋白酶，其分子量为24,000，以合成底物BApNA为底物时的最适pH为10.0。在1 mM DTT存在下，该酶被激活43倍，对丝氨酸（PMSF、TLCK、TPCK）和半胱氨酸（IAA、E - 64）蛋白酶的合成抑制剂不敏感，被STI强烈抑制，并且在酶谱上显示出四条活性带。在PM中，观察到了类胰蛋白酶、类糜蛋白酶、类枯草杆菌蛋白酶和半胱氨酸蛋白酶的活性。未检测到天冬氨酸蛋白酶和金属蛋白酶。在AM中，不寻常的依赖SH的蛋白酶的活性也占主导地位，并且观察到了类糜蛋白酶的活性，但其水平远低于PM。淀粉酶活性在pH 6.0时表现出最佳值，其分布情况则相反。其主要部分（67%）位于AM中。用AM和PM中的蛋白酶处理淀粉酶制剂会使淀粉酶活性降低两倍。中肠内容物的pH值在AM中为6.0 - 7.2，在PM的前半部分为6.4 - 7.6，在PM的后半部分为8.8 - 9.3。因此，AM中的pH值与位于该区室的淀粉酶的最佳pH值相符，但在这样的环境中，包括降解淀粉酶能力在内的蛋白酶活性较低。活跃的蛋白水解发生在PM的后半部分，此处肠道的pH值接近蛋白酶的最佳pH值。
