NMR STUDIES OF CATION TRANSPORT ACROSS BIOMEMBRANES

我们寻求扩展一种新的核磁共振方法，我们 已经发展起来了。它使用了新的移动试剂，顺磁性阴离子，用于 生理碱(Na~+，K~+)和碱性的核磁共振研究 土壤性(Mg++、Ca++)金属水阳离子以及NH4+。它允许 区分细胞内和细胞外离子，从而定量 它们在体内的胞质浓度和物理状态的测定 以及它们以连续方式穿过生物膜的通量。我们 我想沿着几条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核磁共振 细胞内浓度的测量，但我们主要强调的是 探索可以进行的运输测量的种类，以及 证明它们的定量准确性。为此，我们建议；使用 研究离子载体、洗涤剂、 和膜蛋白(如(Na+，K+)ATPase和上皮Na+/H+ 反向转运体)；研究孤立细胞器的自然进出运输 (如线粒体和酵母液泡)；研究生理学和 细胞系统的非生理性流出和流入(如在 人红细胞、培养的心脏细胞和酵母的体外悬浮液)； 以及研究体外组织制剂(如蟾蜍)中的转运 膀胱、灌流鲨鱼直肠腺和灌流大鼠心脏)。 在该提案中，重点是阐明 酵母中NH4+跨简单脂双层转运和阳离子转运 在蟾蜍膀胱和鲨鱼直肠的上皮系统中也有表达 腺体。这些实验大多采用标准方法论 稳定磁性同位素的高场(7T)高分辨核磁共振 的生理阳离子(23Na、39K、25 mg、43Ca和14NH4)中 自然资源丰富。我们的长期目标是证明这一点 方法将成为细胞质定量的常规技术 浓度和跨膜通量及其在生物中的应用 系统将在体内不断增长的曲目中找到自己的位置 局部核磁共振。这项工作涉及到物理和无机两个方面 化学、生物化学、生物物理学和生理学 在对包括高血压在内的多种病理情况的研究中， 情感障碍和癌症。

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.