AMP deaminase (AMPD; EC 3.5.4.6) is a diverse and highly regulated enzyme located at a branchpoint in the adenylate catabolic pathway. AMPD plays a key role during metabolic imbalances of energy supply and demand homeostasis by competing with 5' nucleotidase for available AMP. This role is underscored in striated muscle where inheritance of a prolonged survival in congestive hart failure. These opposing clinical consequences reflect a functional difference in adenylate catabolism in various striated muscles due, in part, to distinct regulatory features of AMPD isoforms. Emerging data show that divergent N-terminal domains in AMPD polypeptides alter physical and functional properties of the enzyme. The long-term objective of this project is to gain a comprehensive understanding of AMPD regulation in striated muscle with an emphasis on the functional significance for divergent N-terminal domains. The continued pursuit of this goal will be accomplished by expressing, isolating an characterizing wild type and genetically modified human AMPD recombinant enzymes that will be used to 1) define the structural bases and functional effects of phosphorylation and phosphoinositides on the catalytic and actomyosin binding properties of human AMPD recombinant isoforms. These analyses will also sere to assess the relationship between these two opposing regulators of AMPD catalytic activity, 2) determine the combined effects of a cassette-type alternative splicing event and a P43L substitution on the human AMPD1 recombinant enzyme. This latter information is central to hypotheses designed to explain clinical outcomes resulting from inheritance of the prevalent AMPD1 mutant allele, and 3) attempt to solve the crystal structure of the human AMPD1 recombinant enzyme. Detailed structural knowledge of AMPD1 will facilitate our understanding of the complex regulation of this enzyme. These combined efforts should provide critical information that may help explain clinical outcomes associated with a prevalent AMPD1 mutant allele.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK050902-11
Application #
6517387
Study Section
Medical Biochemistry Study Section (MEDB)
Program Officer
Mckeon, Catherine T
Project Start
1991-07-01
Project End
2003-06-30
Budget Start
2002-07-01
Budget End
2003-06-30
Support Year
11
Fiscal Year
2002
Total Cost
$208,251
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
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
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

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