PHYSIOLOGY OF INSECT POTASSIUM ION TRANSPORT

已知生电性K+泵位于心尖等离子体上 某些昆虫肠道和感觉上皮细胞的膜。 假设：一个独特的K+-ATPase是这个泵的主要成分，并且 定位于杯状细胞顶膜上的门脉小体。 六爪金龟子的中肠细胞。目标：通过以下方式来描述K+泵 确定K+-ATPase和其他泵成分在分离的 GCAM囊泡和孤立的门静脉小体中。目标：(1)描述 GCAM和溶解形式的K+-ATPase；(2)表征K+ 分离的GCAM囊泡的转运；(3)表征分离的K+ (4)制备抗GCAM蛋白的抗体，用于改良 分离GCAM囊泡和作为泵探针，(5)鉴定抑制剂 K+运输系统。 GCAM囊泡将通过上皮被分离的技术被分离 通过受控的超声波作用层层破碎，然后是膜碎片 是通过差速离心法收集的。K+ATPase将通过以下方法进行检测 孔雀石绿-氨基试剂微量法测定磷酸盐 黑色用于蛋白质测定。膜电位与K+梯度 跨囊泡膜的分布将由 分别标记硫氰酸盐和钾。标记K+泵 蛋白质将被用来分离门脉体。分离细胞系 日本血吸虫杂交瘤细胞株单抗培养液的制备 注射GCAM的小鼠将通过使用GCAM和 泵蛋白作为抗原。氨基甲酸酯、细菌毒素和其他 将测试非神经活性物质抑制K+的特定能力 ATPase。 对于昆虫生理学家来说，独特的K+ATPase可能与 Na+，K+ATPase一直为细胞生理学家所熟知。自.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类病媒(蚊子)幼虫， 特异性K+-ATPase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.