UNIQUE AMINO DOMAINS AND AMP DEAMINASE ISOFORM DIVERSITY

AMP-D是真核生物中普遍存在的一种酶活性。 已经描述了多种异构体，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 联合表达有两种形式。尽管AMP-D的纯化变体存在于 同质异构体，有证据表明， 不同的异构体在体内以异构体的形式存在。一个 表现为运动性肌病的患者中与运动相关的肌病 高度缺乏骨骼肌特异性亚型 AMP-D(即肌腺苷脱氨酶；同型M)具有重要作用 肌细胞能量学中的这种嘌呤代谢酶。反之， 非肌源性AMP-D亚型的功能意义较差 明白了。新近克隆的人不同基因的全长cDNA AMP-D亚型(亚型M和主要活性在肝脏和 许多其他非肌肉组织，同形L)曾戏剧性地预测 氨基末端序列的发散性。这一观察结果表明 对这些不同序列的结构/功能分析可以提供 深入了解AMP-D亚型的特性。建议的实验是 旨在为人类AMP-D生成结构/功能信息 异构体M和L通过：a)正常和 杂合AMP-D多肽(通过切换分歧而产生 氨基末端结构域)由人类表达的DNA产生：1)在 缺乏内源性AMP-D活性的细菌，以及2)在 AMP-D缺乏的酵母菌；b)层析和免疫学 正常和/或异四聚体形成的特征 在细菌和酵母中共表达的杂合AMP-D多肽； C)对已查明的结构突变进行编目和功能测试 多例遗传性Ampd-1基因的分子分析 骨骼肌AMP-D缺乏症。组合在一起，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 多种AMP-D亚型在血管内皮细胞中的功能意义 天哪。

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.