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.
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