PHYSIOLOGY OF INSECT AMINO ACID TRANSPORT

该项目的长期目标是确定其作用机制 电驱动氨基酸、钾离子共转运(Symport) 昆虫，以评估其在K+动态平衡中的作用，并确定符号 抑制剂。工作假说是20个自然发生的 L-氨基酸，从高度碱性的中肠内容物转移到中性 穿过笔刷边界膜。身份识别、隔离和 共转运蛋白一级结构的测定是 达到目标所必需的。分离的刷状边缘膜小泡，BBMV 取食烟草天牛5龄幼虫， 将用于通过快速过滤和荧光研究AA摄取 淬火技术。三个目标自然而然地通向目标。在目标1中 共转运体底物亚群通过阳离子梯度和 用标记AA进行AA-K+吸收的反转运研究 过滤技术。这些技术已经成功地用于 研究鳗鱼BBMV中AA的摄取动力学，但在昆虫研究中是新的。 在AIM中，我们鉴定了3个高亲和力的氨基酸类似物，并将其用于 标记符号蛋白。 该项目将为分离和克隆共同转运蛋白铺平道路。 共转运体基因，并用于确定蛋白质的一级结构。 影响鳞翅目昆虫AA-K+同向转运蛋白的AA类似物 可能被开发为环境安全的虫害防治药剂。这个 极高的管腔pH值(接近11.5)和正PD(接近240 Mv)可能反映在异常的氨基酸阳离子上 与哺乳动物和鸟类不同的是。从幼虫蚊子开始 中肠也有较高的管腔pH，这种AA联合蛋白抑制类似物可能 对蚊子幼虫有选择性毒性，可用于病媒 控制力。

This project's long term goal is to determine the mechanism of action of electrically driven amino acid, potassium ion cotransport (symport) of insects, to assess its role in K+ homeostasis, and to identify symport inhibitors. The working hypothesis is that the twenty naturally occurring L-amino acids, AAs, move from highly alkaline midgut contents to neutral across brush border membranes. Identification, isolation, and determination of the primary structure of the symporter proteins are required to reach the goal. Brush border membrane vesicles, BBMV, isolated from feeding, fifth instar larvae of the tobacco hornworm, Manduca sexta, will be used to study AA uptake by rapid filtration and fluorescence quenching techniques. Three aims lead naturally toward the goal. In Aim 1 symporter substrate sub-groups are identified through cation-gradient and counter-transport studies of AA-K+ uptake using labeled AA, rapid filtration techniques. These techniques have been used successfully to study AA uptake kinetics in eel BBMV but are novel in the study of insects. In Aim 3 high affinity amino acid analogues are identified and used to label symport proteins. The project will pave the way for isolating symporter proteins, for cloning symporter cDNA, and for determining the primary structure of the proteins. AA analogues which interfere with AA-K+ symporter proteins in Lepidoptera may be developed as environmentally safe agents for insect control. The extremely high lumen pH (approaching 11.5) and positive PD (approaching 240 mV) of Lepidopteran midgut may be reflected in unusual AA-cation symporters, unlike those of mammals and birds. Since larval mosquito midguts also have high lumen pH, such AA symport-inhibiting analogues may be selectively toxic to mosquito larvae and be useful in disease vector control.