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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
9R01DK050902-04
Application #
2151988
Study Section
Medical Biochemistry Study Section (MEDB)
Project Start
1991-07-01
Project End
1999-06-30
Budget Start
1995-07-01
Budget End
1996-06-30
Support Year
4
Fiscal Year
1995
Total Cost
Indirect Cost
Name
Medical College of Wisconsin
Department
Biochemistry
Type
Schools of Medicine
DUNS #
073134603
City
Milwaukee
State
WI
Country
United States
Zip Code
53226
Mahnke, Donna K; Sabina, Richard L (2005) Calcium activates erythrocyte AMP deaminase [isoform E (AMPD3)] through a protein-protein interaction between calmodulin and the N-terminal domain of the AMPD3 polypeptide. Biochemistry 44:5551-9
Haas, Amy Louise; Sabina, Richard L (2003) N-terminal extensions of the human AMPD2 polypeptide influence ATP regulation of isoform L. Biochem Biophys Res Commun 305:421-7
Haas, Amy Louise; Sabina, Richard L (2003) Expression, purification, and inhibition of in vitro proteolysis of human AMPD2 (isoform L) recombinant enzymes. Protein Expr Purif 27:293-303
Mahnke-Zizelman, Donna K; Sabina, Richard L (2002) N-terminal sequence and distal histidine residues are responsible for pH-regulated cytoplasmic membrane binding of human AMP deaminase isoform E. J Biol Chem 277:42654-62
Gross, M; Rotzer, E; Kolle, P et al. (2002) A G468-T AMPD1 mutant allele contributes to the high incidence of myoadenylate deaminase deficiency in the Caucasian population. Neuromuscul Disord 12:558-65
Mahnke-Zizelman, D K; Sabina, R L (2001) Localization of N-terminal sequences in human AMP deaminase isoforms that influence contractile protein binding. Biochem Biophys Res Commun 285:489-95
Norman, B; Sabina, R L; Jansson, E (2001) Regulation of skeletal muscle ATP catabolism by AMPD1 genotype during sprint exercise in asymptomatic subjects. J Appl Physiol 91:258-64
Norman, B; Mahnke-Zizelman, D K; Vallis, A et al. (1998) Genetic and other determinants of AMP deaminase activity in healthy adult skeletal muscle. J Appl Physiol 85:1273-8
Mahnke-Zizelman, D K; Tullson, P C; Sabina, R L (1998) Novel aspects of tetramer assembly and N-terminal domain structure and function are revealed by recombinant expression of human AMP deaminase isoforms. J Biol Chem 273:35118-25
Sims, B; Powers, R E; Sabina, R L et al. (1998) Elevated adenosine monophosphate deaminase activity in Alzheimer's disease brain. Neurobiol Aging 19:385-91

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