UNIQUE AMINO DOMAINS AND AMP DEAMINASE ISOFORM DIVERSITY

AMP脱氨酶（AMP-D）是一种普遍存在的真核生物酶。 已经描述了多种同种型，表现出组织特异性和 表达的发育模式，以及大多数组织和细胞 共表达两种形式。 虽然AMP-D的纯化变体以 同源四聚体，证据表明， 不同的同种型在体内结合为异源四聚体。 一个 与运动相关的运动性肌病 严重缺乏骨骼肌特异性同种型 AMP-D（即肌腺苷酸脱氨酶;亚型M）赋予重要作用， 这种嘌呤代谢酶在肌细胞能量学中的作用。 反之， 非肌肉AMP-D亚型的功能意义很差 明白 最近克隆了人全长cDNA， AMP-D同种型（同种型M和在肝脏中表达的主要活性， 许多其他非肌肉组织，同种型L）已经预测了戏剧性的 氨基末端序列的分歧。 这一观察表明， 这些不同序列的结构/功能分析可以提供 深入了解AMP-D异构体特异性特性。拟议的实验是 设计用于生成人类AMP-D的结构/功能信息 亚型M和L通过：a）正常和 杂合AMP-D多肽（通过将趋异的 氨基末端结构域）产生的人cDNA表达：1）在 细菌，其缺乏内源性AMP-D活性，和2）在 AMP-D缺陷型酵母菌株; B）色谱和免疫 正常和/或非正常之间的异源四聚体形成的表征 在细菌和酵母中共表达的杂合AMP-D多肽; c）鉴定的结构突变的编目和功能测试 Ampd-1基因的分子分析，多例遗传性 骨骼肌AMP-D缺乏。 结合起来，从 拟议的研究将提供有价值的结构/功能信息 这将为该项目的长期目标奠定基础，即 阐明多种AMP-D同种型在 伙计

AMP deaminase(AMP-D) is a ubiquitous eukaryotic enzymatic activity. Multiple isoforms have been described, exhibiting tissue-specific and developmental patterns of expression, and most tissues and cells co-express two forms. Although the purified variants of AMP-D exist as homotetramers, evidence has been presented which suggests an ability of different isoforms to associate as heterotetramers in vivo. An associated exercise-induced myopathy in patients presenting with a high-grade deficiency of the skeletal muscle-specific isoform of AMP-D(i.e. myoadenylate deaminase; isoform M) confers an important role to this purine metabolic enzyme in myocyte energetics. Conversely, the functional significance of non-muscle AMP-D isoforms is poorly understood. The recent cloning of full-length human cDNAs for different AMP-D isoforms(isoform M and the major activity expressed in liver and many other non-muscle tissues, isoform L) has predicted dramatic divergence in amino-terminal sequence. This observation suggests that structure/function analyses of these divergent sequences may provide insight into AMP-D isoform-specific properties. Proposed experiments are designed to generate structure/function information for the human AMP-D isoforms M and L through: a)kinetic characterization of normal and hybrid AMP-D polypeptides (generated by switching the divergent amino-terminal domains) produced from human cDNAs expressed: 1) in bacteria, which lack endogenous AMP-D activity, and 2) in AMP-D-deficient strains of yeast; b)chromatographic and immunologic characterization of heterotetramer formation between normal and/or hybrid AMP-D polypeptides co-expressed in bacteria and yeast; c)cataloging and functional testing of structural mutations identified in the Ampd-1 gene by molecular analysis of multiple cases of inherited skeletal muscle AMP-D deficiency. Combined, the data generated from the proposed studies will contribute valuable structure/function information that will lay the foundation for the long-term goal of this project,i.e. elucidating the functional significance of multiple AMP-D isoforms in man.