Presence of chitinase and beta-N-acetylglucosaminidase in the Aedes aegypti A chitinolytic system involving peritrophic matrix formation and degradation
Presence of chitinase and beta-N-acetylglucosaminidase in the Aedes aegypti A chitinolytic system involving peritrophic matrix formation and degradation
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DOI：
10.1016/s0965-1748(02)00112-1
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发表时间：
2002-12-01
影响因子：
3.8
通讯作者：
Pimenta, PFP
中科院分区：
文献类型：
--
作者：
Filho, BPD;Lemos, FJA;Pimenta, PFP
用分光光度法测定特定荧光底物的水解，以及用底物活性-SDS-PAGE凝胶分析法测定埃及伊蚊（Aedes aegypti）肠内几丁质的水解活性，表明几丁质酶和β-N-乙酰氨基葡萄糖苷酶都存在，并具有生理活性。这两种酶甚至存在于未喂食昆虫的肠道中，但在喂食血液或人工无蛋白质饮食后，活性迅速增加。几丁质酶活性主要为“内”型，在36 h达到最大活性，然后在围食基质（PM）降解后下降至非常低的水平。SDS-聚丙烯酰胺凝胶电泳后的几丁质酶检测是一种非常敏感的方法，使我们能够检测到两种不同分子量的几丁质酶在蚊子肠道。一种有效的几丁质酶抑制剂别氨基菌素可抑制蚊肠道中几丁质酶活性对几丁质酶特异性底物的水解。Allosamidin处理导致了非典型厚PM的形成，而外源几丁质酶的加入完全阻断了其形成。这种几丁质分解系统似乎对PM的形成和降解都起作用。由于PM是参与病原体入侵，这些结果是重要的，在促进搜索的机制，可以阻止病原体发展的蚊子载体。(C)2002爱思唯尔科技有限公司版权所有。
Measurement of the hydrolysis of specific fluorogenic substrates by spectrophotometry as well as the substrate activity-SDS-PAGE gel analysis of the chitinolytic activity in Aedes aegypti guts showed that both chitinase and P-N-acetylglucosaminidase are present and physiologically active. Both enzymes were present even in guts from unfed insects, but the activities increased rapidly after feeding on blood or an artificial protein-free diet. Chitinase activity was predominantly of the 'endo'-type, reaching its maximum activity at 36 h and then declining to very low levels after the degradation of the peritrophic matrix (PM). Chitinase assay in gels after SDS-PAGE was a very sensitive method that allowed us to detect two chitinases with distinct molecular weights in the mosquito gut. Hydrolysis of a chitinase-specific substrate by chitinolytic activities in the mosquito guts was inhibited by allosamidin, a potent chitinase inhibitor. Allosamidin treatment led to the formation of an atypical thick PM, while the addition of exogenous chitinase completely blocked its formation. This chitinolytic system appears to operate both on the formation and degradation of the PM. Since the PM is involved in pathogen invasion, these results are important in facilitating a search for mechanisms that can block pathogen development in the mosquito vector. (C) 2002 Elsevier Science Ltd. All rights reserved.