Transport Physiology of Molecular Biology of the Invertebrate Electrogenic 2 Na/1 H Antiporter

小行星9317230 这项调查要求提供资金，研究甲壳类动物胃肠道的离子运输过程，并将其与更彻底调查的哺乳动物进行比较。 在以前的研究中，我们已经确定了一个明显独特的无脊椎动物的肠和肾上皮细胞的刷状缘膜上的产电阳离子交换，这是参与钠和钙的平衡和不同的生理特性从脊椎动物的类似运输蛋白。 本项目将扩展这些早期的研究，使用膜泡技术和分子生物学程序的组合，以表征这种独特的无脊椎动物蛋白质在甲壳类动物的重要生物活性，如离子稳态和蜕皮的功能作用。 为了实现这些目标，将使用标准离心技术从龙虾（Homarus americanus）肝胰腺（后胃的成对憩室）制备刷状缘和基底外侧质膜以及源自与消化和钙调节活性相关的特定细胞内空泡的膜的纯化膜囊泡。 这些囊泡的运输特性将使用放射性同位素和分光光度技术的组合进行研究。 本研究将利用我们先前开发的单克隆抗体作为基因组探针，测定龙虾产电阳离子交换剂的基因核苷酸序列。 从这些生理和分子研究中，我们应该能够澄清这种显然独特的无脊椎动物转运蛋白的功能作用，并将其基因序列与类似但生理上不同的哺乳动物Na +/H+反向转运蛋白亚型进行比较。 ***

9317230 Ahearn This investigation is a request for funds to study ion transport processes of the crustacean gastrointestinal tract and compare them to those of the more thoroughly investigated mammals. In previous studies, we have identified an apparently unique invertebrate electrogenic cation exchanger on the brush border membrane of gut and kidney epithelial cells which is involved in sodium and calcium balance and differs in physiological properties from analogous transport proteins from vertebrates. The present project will extend these early studies using the combination of membrane vesicle techniques and molecular biology procedures to characterize the functional role of this unique invertebrate protein in important biological activities of crustaceans such as ion homeostasis and moltin. To accomplish these objectives, purified membrane vesicles of brush border and basolateral plasma membranes and membranes derived from specific intracellular vacuoles that are associated with digestive and calcium regulatory activities will be prepared from lobster (Homarus americanus) hepatopancreas (paired diverticulum of the posterior stomach) using standard centrifugation techniques. Transport properties of these vesicles will be investigated using a combination of radioisotopic and spectrophotometric techniques. The gene nucleotide sequence of the lobster electrogenic cation exchanger will be determined using our previously developed monoclonal antibody as a probe of the genome. From these physiological and molecular studies we should be able to clarify the functional role(s) of this apparently unique invertebrate transporter and compare its gene sequence with those of analogous, but physiolgically distinct, mammalian Na+/H+ antiporter isoforms. ***