UNIQUE AMINO DOMAINS AND AMP DEAMINASE ISOFORM DIVERSITY

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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.