PHYSIOLOGY OF INSECT AMINO ACID TRANSPORT

昆虫氨基酸运输的生理学

基本信息

批准号：
2065620
负责人：
WILLIAM R HARVEY
金额：
$ 21.04万
依托单位：
TEMPLE UNIV OF THE COMMONWEALTH
依托单位国家：
美国
项目类别：
财政年份：
1990
资助国家：
美国
起止时间：
1990-12-01 至 1998-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/2065620
关键词：
Aedes Insecta Manduca Xenopus oocyte aminoacid transport brush border membrane disease vectors homeostasis immunocytochemistry in situ hybridization insect control ionophores larva membrane transport proteins molecular cloning physiology protein structure function

项目摘要

The LONG TERM OBJECTIVE is to compare the primary structure and functional characteristics of amino acid/alkali metal ion symporter proteins from alkaline midguts of Manduca sexta and Aedes aegypti, to analyses the symporters's roles in chemiosmotically coupled amino acid uptake and to exploit this unusual symport to develop new, environmentally safe mosquitocides. The HYPOTHESIS is that the high alkalinity of caterpillar and mosquito larval midgut is continuously generated by a novel V-ATPase that uses ATP to energize the apical cell membrane by imposing a transmembrane proton electrochemical gradient, delta/mu/H+, lumen positive. The delta/mu/H+ is transformed by a K+/nH+ antiporter to a K+ electrochemical gradient, delta/mu/K+, raising the carbonate-rich lumen pH to >10.5. delta/mu/K+, expressed mainly as delta/psi, chemiosmotically drives amino acid (AA) uptake by alkali adapted AA/K+ symporters (see sketch below). AIM 1 is to characterize glycine/K+ and proline/K+ symporters by rapid filtration studies in brush border membrane vesicles. AIM 2 is to sequence M. sexta symporter cDNA clones, screened by injection of transcribed, sized mRNA fractions from feeding larvae into Xenopus oocytes. AIM 3 is to isolate and sequence symporter cDNA clones from M. sexta and Ae. aegypti midgut cDNA libraries by hybridization with xenic cDNAs encoding known amino acid symporters from vertebrate kidney, brain and other tissues. AIM 4 is to reconstitute purified, recombinant symporters into functional liposomes and characterize them in terms of ionic specificities, kinetic parameters and substrate selectivity, using rapid filtration and fluorescence quenching measurements of amino acid uptake. AIM 5 is to localize amino acid/K+ symporters in midguts from M. sexta and Ae. aegypti by immunocytochemistry and in situ hybridization. SCIENTIFIC MERIT: The use of ATP hydrolysis, chemiosmotically coupled by delta/mu/H+ and delta/mu/K+, to drive amino acid uptake is the first example of such coupling in animal cell plasma membranes. HEALTH RELEVANCE: If larval Ae. aegypti midguts are also energized chemiosmotically by transapical membrane delta/mu/H+ transformed to delta/mu/K+, and if unusual AA/K+ symporters have evolved in the resulting alkaline milieu of disease vector dipteran midguts then agents which interfere with the unusual symporters can be developed as safe mosquitocides.

长期目标是比较主要结构和氨基酸/碱金属离子同向转运蛋白的功能特性来自天蛾和埃及伊蚊的碱性中肠的蛋白质，分析同向转运蛋白在化学渗透偶联氨基酸中的作用吸收并利用这种不寻常的症状来开发新的、对环境安全的杀蚊剂。假设是高毛毛虫和蚊子幼虫中肠的碱度是连续的由一种新型 V-ATP 酶产生，利用 ATP 为顶端细胞提供能量通过施加跨膜质子电化学梯度来形成膜， delta/mu/H+，流明正。 delta/mu/H+ 由 K+/nH+ 转换 K+ 电化学梯度 delta/mu/K+ 的反向转运蛋白，提高富含碳酸盐的管腔 pH 值 >10.5。 delta/mu/K+，主要表示为 delta/psi，化学渗透驱动碱对氨基酸 (AA) 的吸收适应的 AA/K+ 同向转运体（见下图）。目标 1 是通过快速表征甘氨酸/K+ 和脯氨酸/K+ 同向转运蛋白刷状缘膜囊泡的过滤研究。目标 2 是序列 M. sexta 同向转运蛋白 cDNA 克隆，通过注射进行筛选将幼虫喂入非洲爪蟾后转录的、大小一致的 mRNA 片段卵母细胞。目的 3 是从结核分枝杆菌中分离并测序同向转运蛋白 cDNA 克隆。 sexta 和 Ae。通过与异种杂交获得埃及伊蚊中肠 cDNA 文库编码来自脊椎动物肾、脑的已知氨基酸同向转运体的 cDNA 和其他组织。目标 4 是重构纯化的、重组的将转运蛋白转运至功能性脂质体中，并根据以下方面对它们进行表征离子特异性、动力学参数和底物选择性，使用氨基酸的快速过滤和荧光猝灭测量吸收。目标 5 是定位中肠中的氨基酸/K+同向转运蛋白 M. sexta 和 Ae。通过免疫细胞化学和原位检测埃及伊蚊杂交。科学价值：利用 ATP 水解，通过化学渗透耦合 delta/mu/H+ 和 delta/mu/K+，以驱动氨基酸摄取为先动物细胞质膜中这种偶联的例子。健康相关性：如果是 Ae 幼虫。埃及伊蚊的中肠也充满活力经心尖膜通过化学渗透将 delta/mu/H+ 转化为 delta/mu/K+，如果不寻常的 AA/K+ 同向转运蛋白在由此产生的疾病媒介双翅目中肠的碱性环境然后是药剂干扰异常同向转运体的药物可以被开发为安全的杀蚊剂。