Electrophoretic separation and identification of phenoloxidases in hemolymph and midgut of adult Anopheles stephensi mosquitoes
Electrophoretic separation and identification of phenoloxidases in hemolymph and midgut of adult Anopheles stephensi mosquitoes
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DOI：
10.2307/3284247
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发表时间：
1997-08-01
影响因子：
1.3
通讯作者：
Vanderberg, JP
中科院分区：
文献类型：
--
作者：
Sidjanski, S;Mathews, GV;Vanderberg, JP
按蚊对入侵的疟原虫的排斥反应机制是由酚氧化酶介导的蚊子中肠上皮中动合子的黑化组成。血淋巴与中肠酚氧化酶在这种排斥机制中的相对作用尚不清楚。采用非变性凝胶电泳结合酶谱分析的方法，对斯氏按蚊中肠和血淋巴的酚氧化酶同工酶进行了分离和鉴定。两个地点的同工酶具有明显不同的电泳特征。血淋巴有2条酚氧化酶阳性带，这两条带都是氧化单酚和邻二酚底物的双功能分子。中肠提取物有3条带，迁移速度比血淋巴快。这些中肠带没有可检测的monophenoloxidase活性，他们拥有，但是，广谱的二酚氧化酶的活性，在其氧化邻和对-二酚底物的能力，以及漆酶底物alcingaldazine。通过对螯合剂托酚酮抑制的不敏感性，可以将2条迁移最快的中肠PO带与迁移更慢的带区分开来。血淋巴和中肠酚氧化酶在蚊虫防御入侵性寄生虫中的相对作用是未来有待解决的问题。
Anopheles mosquitoes frequently respond to invading malaria parasites with a rejection mechanism consisting of phenoloxidase-mediated melanization of ookinetes in the mosquito midgut epithelium. The relative roles of hemolymph vs. midgut phenoloxidase in this rejection mechanism is unclear. We have separated and identified phenoloxidase isozymes from midgut and hemolymph of Anopheles stephensi by native gel electrophoresis followed by zymography. The isozymes from the 2 sites had distinctively different electrophoretic characteristics. Hemolymph possessed 2 phenoloxidase-positive bands, both of which were bifunctional molecules that oxidized monophenol as well as o-diphenol substrates. Midgut extract possessed 3 bands that migrated more rapidly than those of the hemolymph. None of these midgut bands had detectable monophenoloxidase activity; they possessed, however, a broad spectrum of diphenoloxidase activity in their ability to oxidize both o- and p-diphenol substrates, as well as the laccase substrate syringaldazine. The 2 most rapidly migrating midgut PO bands could be distinguished from the more slowly migrating band through their insensitivity to inhibition by the chelating agent tropolone. A question to be resolved in the future relates to the relative roles of hemolymph vs. midgut phenoloxidase in mosquito defense against invasive parasites.