PHYSIOLOGY OF INSECT POTASSIUM ION TRANSPORT

生电钾泵定位于心尖部血浆 某些昆虫肠和感觉上皮细胞的膜。 假设：一个独特的K+ ATP酶是这个泵的主要组成部分， 定位于门体的杯状细胞顶膜（GCAM）上， 中肠细胞。 目标：通过以下方式表征K+泵 确定K+ ATP酶和其他泵组分在分离的 GCAM囊泡和分离的门户体中。 目的：（1）表征 GCAM和溶解态K+ ATP酶;（2）研究K + ATP酶的特性 通过分离的GCAM囊泡转运;（3）表征分离的K+ （4）产生针对GCAM蛋白的抗体，以改善 分离GCAM囊泡并作为泵探针，（5）鉴定抑制剂 K+运输系统 GCAM囊泡将通过以下技术分离： 通过受控的超声处理分层破坏， 通过差速离心收集。 K+ ATP酶将通过 微量孔雀绿绿色法测定磷酸盐和氨基 黑色用于蛋白质测定。 膜电位和K+梯度 将通过以下物质的分布来确定 放射性标记的硫氰酸盐和钾。 标记的K+泵 蛋白质将用于分离转运体。 分离细胞系 制备单克隆抗体的杂交瘤培养液， 用GCAM注射的小鼠将通过ELISA试验使用GCAM筛选， 泵蛋白作为抗原。 氨基甲酸酯、细菌毒素和其他 将测试非神经活性药物抑制K+的特异性能力 ATP酶 对昆虫生理学家来说，独特的K+ ATP酶可能与 Na ~+，K ~+ ATP酶是细胞生理学家熟知的。 以来 胃肠道形式的酶可能局限于中肠， 某些鳞翅目害虫和双翅目疾病媒介（蚊子）幼虫， 特异性K+ ATP酶抑制剂可能是安全的杀虫剂。

An electrogenic K+ pump is known to be localized on the apical plasma membrane of certain insect intestinal and sensory epithelial cells. Hypothesis: a unique K+ ATPase is the principal component of this pump and is localized in portasomes on the goblet cell apical membrane (GCAM) of midgut cells in Manduca sexta. Goal: to characterize the K+ pump by determining the role of the K+ ATPase and other pump components in isolated GCAM vesicles and in isolated portasomes. Aims: (1) to characterize the K+ ATPase both in GCAM and in solubilized form; (2) to characterize K+ transport by isolated GCAM vesicles; (3) to characterize isolated K+ portasomes; (4) to produce antibodies against GCAM proteins for improved isolation of GCAM vesicles and as pump probes, (5) to identify inhibitors of the K+ transport system. GCAM vesicles will be isolated by techniques in which the epithelium is disrupted in layers by controlled sonication and then membrane fragments are collected by differential centrifugation. K+ ATPase will be assayed by a micro method using malachite green for phosphate determination and amido black for protein determination. Membrane potential and K+ gradients across vesicle membranes will be determined by distribtution of radiolabeled thiocyanate and potassium, respectively. Labeled K+ pump proteins will be used to isolate portasomes. To isolate cell lines producing monoclonal antibodies culture fluid of hybridomas prepared from mice injected with GCAM will be screened by an ELISA assay using GCAM and pump proteins as antigen. Carbamates, bacterial toxins, and other nonneuroactive agents will be tested for specific ability to inhibit K+ ATPase. The unique K+ ATPase may be as significant to insect physiologists as the well known Na+, K+ ATPase has been to cell physiologists. Since the gastrointestinal form of the enzyme may be restricted to the midgut of certain lepidopterous pest and dipterous disease vector (mosquito) larvae, specific K+ ATPase inhibitors may be safe insecticides.