NMR STUDIES OF CATION TRANSPORT ACROSS BIOMEMBRANES

阳离子跨生物膜传输的核磁共振研究

• 批准号: 3280742
• 负责人: CHARLES S. SPRINGER
• 金额: $ 11.41万
• 依托单位: STATE UNIVERSITY OF NEW YORK AT ALBANY
• 依托单位国家: 美国
• 财政年份: 1984
• 资助国家: 美国
• 起止时间: 1984-04-01 至 1987-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3280742
• 关键词: adenosinetriphosphatase; ammonium compounds; cations; erythrocytes; heart cell; human tissue; ionophores; membrane model; membrane permeability; microsomes; nuclear magnetic resonance spectroscopy; reagent /indicator; renal medulla; saltwater environment; stable isotope; stoichiometry; urinary bladder; yeasts

We seek to expand a new nuclear magnetic resonance (NMR) approach which we have developed. It employs new shift reagents, paramagnetic anions, for the study of the NMR of the physiological alkali (Na+,K+) and alkaline earth (Mg++, Ca++) metal aquo cations as well as NH4+. It allows discrimination of intra- and extracellular ions and thus quantitative measurement of their cytoplasmic concentrations and physical state in vivo and of their fluxes across biological membranes in a continuous manner. We want to expand this approach along a number of parallel lines. These include testing new, more effective, shift reagents and quantifying the NMR measurements of intracellular concentrations, but our main emphasis is to explore the kinds of transport measurements which can be made and to demonstrate their quantitative accuracy. To this end, we propose; to use vesicles to study effluxes and influxes induced by ionophores, detergents, and membrane proteins (such as (Na+, K+)ATPase, and epithelial Na+/H+ antiporter); to study natural transport into and out of isolated organelles (such as mitochondria and yeast vacuoles); to study physiological and non-physiological effluxes and influxes of cellular systems (such as in vitro suspensions of human erythrocytes, cultured heart cells, and yeasts); and to study transport in in vitro tissue preparations (such as toad urinary bladder, perfused shark rectal gland and perfused rat hearts). Emphasis is given in the proposal to the elucidation of the mechanisms of NH4+ transport across simple lipid bilayers and cation transport in yeast and in the epithelial systems of toad urinary bladder and shark rectal gland. These experiments mostly employ the standard methodology of high-resolution NMR, at high fields (7T), of the stable magnetic isotopes of the physiological cations (23Na, 39K, 25Mg, 43Ca, and 14NH4) present in natural abundance. Our long-term objective is to demonstrate that this approach will become a routine technique for quantitating cytoplasmic concentrations and transmembrane fluxes and that its application to living systems will find its place in the growing repertoire of in vivo and topical NMR. This work involves aspects of physical and inorganic chemistry, biochemistry, biophysics, and physiology and has ramifications in the study of a number of pathological conditions including hypertension, affective disorders, and cancer.

我们寻求扩展一种新的核磁共振（NMR）方法， 已经发展。 它采用了新的位移试剂，顺磁性阴离子， 生理碱（Na+，K+）和碱金属离子的核磁共振研究 土（Mg++，Ca++）金属水阳离子以及NH 4+。 它允许 区分细胞内和细胞外离子， 测量其细胞质浓度和体内物理状态 以及它们以连续的方式穿过生物膜的通量。 我们 我想沿着一些平行线扩展这种方法。 这些 包括测试新的，更有效的，位移试剂和量化NMR 细胞内浓度的测量，但我们的主要重点是 探索可以进行的各种运输测量， 证明其定量准确性。 为此，我们建议： 研究离子载体，去污剂， 膜蛋白（如（Na+，K+）ATP酶和上皮细胞Na+/H+ 反向转运蛋白）;研究自然运输进出孤立的细胞器 (such如线粒体和酵母空泡）;研究生理和 细胞系统的非生理流出和流入（例如， 人红细胞、培养的心脏细胞和酵母的体外悬浮液）; 并在体外组织制备物（如蟾蜍）中研究转运 膀胱、灌注的鲨鱼直肠腺和灌注的大鼠心脏）。 在建议中强调阐明的机制， 酵母中NH_4 ~+跨简单脂双层转运和阳离子转运 在蟾蜍膀胱和鲨鱼直肠上皮系统中 腺。 这些实验大多采用标准的方法， 稳定磁性同位素的高分辨率NMR，在高场（7 T） 生理阳离子（23 Na，39 K，25 Mg，43 Ca和14 NH 4）存在于 自然丰富。 我们的长期目标是证明， 方法将成为定量细胞质的常规技术， 浓度和跨膜通量及其在生活中的应用 系统将在不断增长的体内和 局部NMR。 这项工作涉及物理和无机方面 化学、生物化学、生物物理学和生理学， 在包括高血压在内的许多病理状况的研究中， 情感障碍和癌症。